Structural elucidation of a common architecture of the nuclear pore
complex and COPIl vesicle coats
By
MASSACHUSETTS INSTRV11Ji
OF TECHNOLOGY
Stephen Graf Brohawn
MAR 17 2010
B.S. Biochemistry
University of Delaware, 2004
LIBRARIES
Submitted to the Department of Biology in partial fulfillment of the requirements
for the degree of
ARCHIVES
Doctor of Philosophy
at the
Massachusetts Institute of Technology
June 2010
@2010 Stephen Graf Brohawn. All Rights Reserved.
The author hereby grants to MIT permission to reproduce and to distribute
publicly paper and electronic copies of this thesis document in whole or in part in
any medium now known or hereafter created.
Signature of Author:
Department of Biology
.
Certified by:_
~February
10, 2010
Thomas U. Schwartz
Associate Professor of Biology
Thesis Supervisor
Accepted by:_
Stephen P. Bell
Professor of Biology
Co-Chair, Biology Graduate Committee
2
Structural elucidation of a common architecture of the nuclear pore
complex and COPII vesicle coats
By
Stephen Graf Brohawn
Submitted to the Department of Biology in partial fulfillment of the
requirements for the degree of Doctor of Philosophy at the
Massachusetts Institute of Technology
Abstract
Nuclear pore complexes (NPCs) are massive protein assemblies that perforate the
nuclear envelope and form the exclusive passageway for nucleocytoplasmic transport.
NPCs play critical roles in molecular transport and a myriad of other cellular processes.
Elucidation of the structure of the NPC is thus expected to provide important insight into
cell biology. In this thesis, I investigate the structure of a key subcomplex of the NPC
and discuss the evolutionary relationship between the NPC and COPIl vesicle coats it
illustrates.
The NPC is a modular assembly, with a stable structural scaffold supporting dynamically
attached components. The structural scaffold is constructed from multiple copies of the
Y-shaped complex and the Nic96 complex. We solved the crystal structure of the
heterodimeric Nup85-Sehl module that forms a short arm in the Y complex. Nup85 is
found to contain a conserved fold, the ancestral coatomer element 1 (ACE1), also
present in three other components of the NPC and in the COPI vesicle coat, providing
structural evidence of coevolution from a common ancestor.
Sec3l ACE1 units interact to form edge elements in the COPI lattice. Using structural
knowledge of this edge element, we identified corresponding interactions between ACE1
proteins Nup84 and Nup145C in the NPC. We solved the crystal structure of the
heterotrimeric Nup84-Nupl 45C-Secl 3 module that forms the top of the long arm in the
Y complex. The heterotypic ACE1 interaction of Nup145C and Nup84 is analogous to
the homotypic Sec31 edge element interaction in the COPIl coat.
From these and other structures, we assemble a near complete structural model of the Y
complex. Further, based on the demonstrated relationship with the COPIl coatomer
lattice, we propose a lattice model for the entire NPC scaffold. The common architectural
principles of the edge elements in the NPC and COPI lead us to predict that Y
complexes will be arranged as struts in the NPC lattice. In this manner, Nup84-Nup145C
edge elements are arranged parallel to the transport axis to stabilize the positively
curved nuclear envelope. From a lattice model of the NPC follow hypotheses for how
other components are integrated into and function within the NPC.
Thesis Supervisor: Thomas U. Schwartz
Title: Pfizer-Laubach Career Development Associate Professor of Biology
4
Acknowledgements
I am fortunate enough to have had an exceptionally rewarding and fun time in
graduate school and for that I have a lot of people to thank.
First, I would like to thank my advisor Thomas Schwartz for giving me the
opportunity to work and learn in his lab. I feel so lucky to have been able to
experience his unwavering support, infectious curiosity and excitement, and real
passion for science in the lab for the past five years. He created and fosters a
fantastic environment for work, science, and everything else, and for that I am
very grateful. I would also like to thank Bob Sauer and Mike Yaffe for being on
my thesis committee. Their insights and advice offered at every meeting have
been a great source of direction and have contributed substantially to my work. I
would especially like to thank them for being so accessible throughout the entire
process. I also thank Cathy Drennan and Steve Harrison for joining my
committee. I am greatly appreciative to these scientists that I respect so much for
helping me learn to be one.
Thanks go to everyone who is in and has been in the Schwartz lab. I would
especially like to thank the other graduate students in the lab; James Partridge
and James Whittle, who made joining a new lab seem a lot less crazy and a lot
more fun, and Nina Leksa, who has been just fantastic to work with on a lot of the
projects in this thesis since she joined. Thanks to the first post docs, Thomas
Boehmer and then Sandra Jeudy, for showing me a lot and having a lot of fun as
well. Thanks to the rest of the current group - Silvija Bilokapic, Brian Sosa,
Raven Ready, and Alexander Ulrich - and to all the other people who have left for
making the lab a great place to be. Thanks also to Eric Spear for tons of help.
Special thanks go to Lena Karotki, Sabin Mulepati, Glenna Wink, and Marko
Gogola for working with me and in the end teaching me more than I taught them!
Lastly, thanks go to my family and friends outside the lab. Thanks to Mom and
Dad for giving me the opportunity to be here and for always encouraging me to
get involved in the things I was interested in. Thanks also to the rest of my family
and friends, especially my brothers Dan, Dave, and Mike, for perspective and
support. And finally thanks to Katie, for being with me the whole way through.
6
Table of Contents
Title Page
1
Abstract
3
Acknowledgements
5
Table of Contents
7
List of Figures
10
List of Tables
13
Chapter one: The nuclear pore complex has entered the atomic age
14
Abstract
Introduction
Overall structure
Modularity
Protein composition
Subcomplexes
Dynamics
Domain architecture
FG-repeats
Coiled-coils
p-propellers
a-helical domains
ACE1 domains
Assembly
Comparison to vesicle coats
Outlook
Acknowledgements
Chapter two: Structural evidence for common ancestry of the nuclear
pore complex and vesicle coats
Abstract
Introduction
Results
Discussion
Methods
Protein production and purification
Crystallization
Structure determination
Analytical gel filtration
15
15
16
19
20
22
24
24
25
25
26
29
32
33
36
39
40
41
42
42
43
65
74
74
76
76
77
Analytical ultracentrifugation
Isothermal titration calorimetry
CD spectroscopy
In vivo localization experiments
Yeast plasmid construction
Strain construction
Cell fractionation
Acknowledgements
Chapter three: The structure of the scaffold nucleoporin Nup120 reveals
a new and unexpected domain architecture
Abstract
Introduction
Results
Structure determination
Crystal structure of Nup120
The main crystal contact is formed by a domain swap
Conservation of Nup120 and comparison to the human
ortholog Nup160
The C terminus of Nup120 directly binds Nup145C and
Nup85
Without its C-terminal domain Nup120 does not properly
Localize to the NPC
Nup120 is topologically different from other scaffold
Nucleoporins
Discussion
Nup120 in the context of the NPC scaffold
Experimental procedures
Protein expression and purification
Protein crystallization
Structure determination
Analytical size exclusion chromatography
Yeast strain construction
Fluorescence microscopy
Accession numbers
Acknowledgements
Chapter four: Molecular architecture of Nup84*Nupl45C*Secl3 edge
element in the nuclear pore complex lattice
Abstract
Introduction
Results
78
79
79
79
80
80
81
81
83
84
84
87
87
88
90
91
93
95
97
101
101
104
104
105
106
106
106
107
107
107
108
109
109
112
Structure of Nup84-Nup145C-Sec13 trimeric
complex
ACE1 nucleoporins Nup84 and Nup145C interact
crown to crown
Structural evidence for lattice model of the NPC
Comparison of edge elements in NPC and COPII
coat
Discussion
Methods
Construct generation
Protein production and purification
Crystallization
Data collection and structure determination
Structure analysis
Acknowledgements
112
116
121
123
126
132
132
132
132
133
135
135
Chapter five: Extending the lattice model of the nuclear pore complex
136
From A to Y: a complete Y complex structure
Beyond the Y: incorporation of other nucleoporins into the NPC
lattice
@-propellers and vertex elements in the NPC lattice
a-helical nucleoporins in the NPC lattice
ACE1 nucleoporins
Nupl33/Nup170 and other helical nucleoporins adaptors/tethers in the NPC?
Overall features
Flexibility of the NPC
Lattice architecture and membrane curvature
Summary
137
Appendix: A lattice model for the nuclear pore complex
Abstract
Discussion
Acknowledgements
References
139
139
142
142
143
145
145
145
148
149
150
150
155
156
List of Figures
Figure 1.1
Overall structure of the nuclear pore complex
17
Figure 1.2
Schematic representation of the modular assembly
of the NPC
20
Figure
Inventory of the NPC
22
Figure
Structures of nucleoporins
28
Figure
The Ancestral Coatomer Element ACE1
31
Figure
Assembly models for the NPC
34
Figure
Predicted lattice-like arrangement of Y complexes in the
NPC
38
Figure 2.1
Structure of the Nup85-Sehl complex
45
Figure 2.2
Arrangement of two Nup85-Sehl heterodimers in the
asymmetric unit
47
Nup85-Sehl is a dimer in solution as determined by
analytical ultracentrifugation
48
Nup85*Sehl is a dimer in solution as determined by
size exclusion chromatography
49
Comparison of Nup85-Sehl and Nup145C-Sec13 and
identification of the Nup84-Nup145C crown-crown
binding interface
50
Nupl20 binds Nupl45C and Nup85 via their tail
modules
52
A single point mutation in the predicted interaction helix
of the Nup145C tail module disrupts Nup120 binding
54
Surface mutation of the Nup145C and Nup84 crowns
does not negatively affect protein stability
55
Elimination of the Nup84 binding site on Nup145C
results in nuclear pore assembly defects in vivo
57
Figure 2.3
Figure 2.4
Figure 2.5
Figure 2.6
Figure 2.7
Figure 2.8
Figure 2.9
Figure 2.10
Growth analysis of yeast strains
Figure 2.11
Nup133-GFP and Nup84-GFP become more soluble in
the Nup145-ELIEA strain
Figure 2.12 Architecture of ACE1
Figure 2.13
Superposition ACE1 modules
Figure 2.14
Surface mutations of the Nup145C and Nup84 crowns
Figure 2.15
Sequence alignments
Figure 2.16
Current model for the Y complex and the
structural scaffold of the nuclear pore complex
Figure 3.1
Overall topology of Nup120
Figure 3.2
Crystal contacts between two symmetry-related
molecules
Figure 3.3
Surface conservation and electrostatics of Nup120
Figure 3.4
The C-Terminus of Nup120 Is necessary for binding
Nup85-Sehl and Nup145C-Sec13
Figure 3.5
Nup120 1. 757 does not localize to the nuclear envelope
Figure 3.6
Nup120 is composed of a combined P-propellera-helical domain distinct from ACE1- p-propeller
Figure 4.1
Structure of Nup84*Nupl45C-Secl3
Figure 4.2
Electron density for the Nup84-Nup145C-Secl 3 model
Figure 4.3
Nup84 is an ACE1-containing protein
Figure 4.4
The crown-crown interaction of Nup84-Nup145C is
analogous to that of Sec3l *Sec31
119
The hydrophobic and highly conserved
Nup84-Nup145C interface
120
The Nup84*Nupl45C*Secl3 edge element sterically
clashes with a proposed "fence-like" pole in the NPC
123
Figure 4.5
Figure 4.6
Figure 4.7
Figure 4.8
Figure 4.9
Figure A.1
Comparison of edge elements in the NPC and
COPIl lattices
125
Nup84*Nupl45C is a membrane curvature-stabilizing
edge element in the NPC lattice
128
The Y complex does not display a positively
charged surface characteristic of membrane interaction
131
A lattice model of the NPC
153
List of Tables
Table 2.1
Data collection and refinement statistics
44
Table 2.2
Strains used in this study
81
Table 3.1
Data collection and refinement statistics
88
Table 4.1
Data collection and refinement statistics
113
Chapter one
The nuclear pore complex has entered the atomic age
This chapter was previously published as Brohawn, S.G.*, Partridge, J.R.*,
Whittle, J.R.R.* & Schwartz, T.U. The nuclear pore complex has entered the
atomic age. Structure 17, 1156-1168 (2009).
*These authors contributed equally to this work.
S.G.B., J.R.P., and J.R.R.W. and T.U.S. prepared figures and wrote the
manuscript; T.U.S. advised on all aspects of the project and wrote the
manuscript.
Abstract
Nuclear pore complexes (NPCs) perforate the nuclear envelope and represent
the exclusive passageway into and out of the nucleus of the eukaryotic cell. Apart
from their essential transport function, components of the NPC have important,
direct roles in nuclear organization and in gene regulation. Due to its central role
in cell biology, it is of considerable interest to determine the NPC structure at
atomic resolution. The complexity of these large, 40-60 MDa protein assemblies
has for decades limited such structural studies. More recently, exploiting the
intrinsic modularity of the NPC, structural biologists are making progress toward
understanding this nanomachine in molecular detail. Structures of building
blocks of the stable, architectural scaffold of the NPC have been solved, and
distinct models for their assembly proposed. Here we review the status of the
field and lay out the challenges and the next steps toward a full understanding of
the NPC at atomic resolution.
Introduction
The hallmark of eukaryotic cells is an elaborate endomembrane system that
creates membrane-enclosed organelles. The nucleus is the most prominent
organelle, as it harbors the genetic material of the cell. NPCs are the only
gateways to the nucleus and reside in circular openings in the nuclear envelope,
where the inner nuclear membrane (INM) and outer nuclear membrane (ONM) of
the nuclear envelope (NE) are fused. NPCs are among the largest multiprotein
assemblies in the quiescent cell and were first described 50 years ago with
electron microscopy (Watson, 1959). Here we provide a snapshot on the status
of the structural characterization of the nuclear pore complex - the results of a
truly multi-disciplinary approach. For general reviews the reader is also referred
to (D'Angelo and Hetzer, 2008; Lim et al., 2008; Tran and Wente, 2006), for the
mechanism of NPC assembly to (Antonin et al., 2008), and for nucleocytoplasmic
transport of proteins and RNAs to (Carmody and Wente, 2009; Cook et al., 2007;
Kohler and Hurt, 2007; Pemberton and Paschal, 2005; Stewart, 2007; Weis,
2003). The role of the NPC in gene regulation and nuclear organization is
addressed in (Akhtar and Gasser, 2007; Heessen and Fornerod, 2007).
Overall structure
Electron microscopy has been the best technique to observe the overall structure
of the NPC. A variety of cell types from different organisms have been imaged. In
its internal symmetry, shape, and size the NPC seems conserved throughout
evolution, though at the molecular level there are differences, as noted below. In
internal symmetry, the first electron micrographs of the NPC showed that it forms
an octagonal ring whose central channel is less electron dense then the eight
lobes that surround it. In shape, scanning electron microscopy experiments
(SEM) have recorded some of the most stunning NPC images (Figure 1.1). While
the architectural core is grossly symmetric about the plane of the membrane, the
peripheral components on the nuclear and cytoplasmic faces are distinct. These
peripheral components recapitulate the eightfold symmetry about the transport
axis exhibited by the architectural core. On the cytoplasmic side, eight knobs,
thought to be attachment sides for fibrilic extensions, are visible in NPCs from
multicellular species (Kiseleva et al., 2000). In yeast, these features are less
pronounced, but are likely also present (Kiseleva et al., 2004). On the
nucleoplasmic side, a ring termed the nuclear basket is suspended from eight
filaments that join it to the NPC. Lastly, in size, the overall and central channel
diameters of the NPC appear to be similar in all eukaryotes, about 90-120nm and
50nm respectively (Akey and Radermacher, 1993; Beck et al., 2007; Fahrenkrog
et al., 2000; Frenkiel-Krispin et al., 2009; Hinshaw et al., 1992; Stoffler et al.,
2003). However, there is still considerable uncertainty about the height,
determined to be between ~30-95nm (Alber et al., 2007b; Elad et al., 2009).
A
Xenopus
laevis
Drosophila Saccharomyces
melanoaaster cerevisiae
cytoplasmic side
nucleoplasmic side
side view
cytoplasmic view
Figure 1.1 - Overall structure of the nuclear pore complex
(A) Representative micrographs of NPCs from diverse eukaryotes obtained by
scanning electron microscopy. The distinct surface features that define
cytoplasmic and nucleoplasmic faces of the NPC are conserved, so are the
overall dimensions in the plane of the nuclear envelope. Scale bar indicates
100nm.
(B) Cryo-electron tomographic (cryo-ET) reconstruction of the human NPC. The
nuclear basket structure and the cytoplasmic extensions are omitted for clarity.
The central eightfold rotational symmetry is clearly visible. A comparison
between cryo-ET reconstructions of NPCs from diverse species reveals
substantial differences in the overall height (Elad et al., 2009).
Further work has led to progressively more detailed reconstructions of the NPC.
Cryo-electron microscopy that relies on averaging images from many NPCs has
been employed to study the core NPC structure, the part that spans the distance
between the faces of INM and ONM (Akey and Radermacher, 1993; Hinshaw et
al., 1992). These studies have shown that the scaffold ring structure - the
electron dense material near the nuclear membrane - has alternating thicker and
thinner regions, hence it is often called the spoke ring ((Akey and Radermacher,
1993; Hinshaw et al., 1992) Due to the confusing nature of this terminology, in
this thesis, "scaffold ring" will be used and what are referred to as "spokes" in the
literature will be referred to as scaffold segments). The scaffold structure appears
to penetrate the pore membrane to also form a perinuclear ring structure. Using
cryo-electron tomography, the best pictures of complete NPCs have been
achieved, extending even to a resolution of ~ 6 nm (Beck et al., 2007; Elad et al.,
2009; Frenkiel-Krispin et al., 2009). With this technique, details of the ring
structures become apparent. The scaffold can be divided into three main ring
elements: a central scaffold ring sandwiched between a cytoplasmic ring and a
nucleoplasmic ring. The rings appear to float on top of one another or to be thinly
connected, indicating that material connecting these rings is less electron-dense.
Alternatively, this may be due to technical difficulties, such as the 'missing cone'
problem or poor resolution in the Z-direction. Recent improvement in the
resolution of a Xenopus NPC reconstruction has further delineated the scaffold
ring into two concentric rings in the plane of the NE connected with a high density
mass perpendicular to the NE (Frenkiel-Krispin et al., 2009).
The central transport cavity of the nuclear pore complex shows no distinct
structural features, consistent with the perception that it is filled by an aqueous
meshwork formed by natively unfolded FG-domains, which are long polypeptide
sequences found in several nucleoporins that contain phenylalanine-glycine (FG)
repeats but are otherwise hydrophilic. These extensions are thought to form a
distinct, semi-permeable environment that prevents the diffusion of large
molecules, unless they are bound to nuclear transport factors (NTRs) that
facilitate entry into this central cavity. NTRs recognize export- or import-specific
signal sequences on cargo molecules and interact directly with FG-nups to
facilitate transport. Directional specificity of transport is accomplished by the
small GTPase Ran-mediated regulation of cargo-NTR interactions in a
nucleotide-bound state-dependant fashion. A gradient of Ran nucleotide binding
states is maintained in turn by the cytoplasmic localization of the Ran GTPaseactivating protein (RanGAP) and nuclear localization of the Ran guaninenucleotide exchange factor (RCC1).
In addition to the central channel, the scaffold itself likely harbors additional
peripheral channels. The scaffold ring appears porous in cryo-EM/-ET structures,
with gaps of - 9 nm diameter close to the NE membrane (Hinshaw et al., 1992;
Stoffler et al., 2003). Peripheral channels have been discussed in several
studies, and were postulated to transport small proteins and ions (Kramer et al.,
2007). It is unclear, however, how this type of transport could be restricted to the
peripheral channels, when the central channel could allow it as well.
Alternatively, it also has been suggested that the peripheral channels transport
membrane proteins destined for the INM. These are inserted into the ER
membrane following translation and stay membrane-anchored until they reach
their final destination (the ER, ONM, and INM are all contiguous). Perhaps these
membrane proteins pass the NPC into the nucleus via these peripheral channels
(Powell and Burke, 1990; Zuleger et al., 2008). The nucleoplasmic domains of
INM proteins are limited in size to ~ 40 kDa, small enough to fit into the cavities
of the observed channels.
While the general NPC architecture is well established, the cryo-EM/-ET
structures do not permit the assignment of individual proteins, since their
boundaries are not visible at this resolution. For this, higher resolution methods
are required.
Modularity
A characteristic of the NPC is its high degree of modularity, which manifests itself
at several levels. First, the NPC is organized around a central eightfold rotational
symmetry. Second, only -30 nucleoporins, composed of a limited set of domain
topologies, build the NPC. Third, nucleoporins have various dwell times at the
NPC, with only a fraction being stably attached at all times. And finally, the
stably attached nucleoporins are arranged into subcomplexes, each of which
assembles in multiple copies to build the entire NPC (Figure 1.2). This modularity
is the basis for approaching structural determination of the assembly at atomic
resolution (Schwartz, 2005).
S.cerevisiae
Ycomplex members:
Nup84, Nup85, Nup120, Nup133, Nup145C, Sec13, Seh1
Metazoa
Ycomplex members:
Nupl07, Nup85, Nup160, Nup133, Nup96, Sec13, Seh1,
Nup37, Nup43, ELYS
Figure 1.2 - Schematic representation of the modular assembly of the NPC
The NPC is built from ~30 nucleoporins, organized in a small set of
subcomplexes. The cartoon shows the major subcomplexes that make up the
lattice-like scaffold (blue colors), the membrane-attachment (yellow), and the FGnetwork (grey) of the NPC. S.cerevisiae components on the left, metazoa, with
specific additional components, on the right. A few peripheral nups are left out for
clarity. Simplified representation, connections are not to be taken literally and box
sizes are not proportional to molecular weights.
Protein composition
Three studies, using S. cerevisiae (Rout et al., 2000), the trypansome T. brucei
(DeGrasse et al., 2009), or rat hepatocytes (Cronshaw et al., 2002) as starting
material, focused on determining the complete inventory of nucleoporins. In each
study, cell extracts were enriched for NPCs by fractionation techniques and the
purified proteins were then analyzed by mass-spectrometry. The set of proteins
found in each organism is largely identical and comprises ~ 30 different gene
products. The nucleoporins can be broadly classified into three categories
(Figure 1.3). -10 contain disordered N- and/or C-terminal regions that are rich in
phenylalanine-glycine (FG) repeats. These FG-repeat regions emanate into and
form the transport barrier in the channel of the NPC. -15 nucleoporins have
distinct architectural functions and form the NPC scaffold structure. Three
nucleoporins have transmembrane domains and anchor the NPC in the circular
openings in the NE. Immunogold-labeling of all nucleoporins shows that the
majority of the nups, notably scaffold nucleoporins, are symmetrically localized
around a two-fold symmetry axis in the plane of the NE, perpendicular to the
eightfold rotational symmetry about the main transport channel (Rout et al.,
2000). Based on simple hydrodynamic and volumetric calculations the size of the
NPC was estimated to range from 66 MDa in S. cerevisiae (Rout and Blobel,
1993) to 125 MDa in vertebrates (Reichelt et al., 1990). Calculations based on
the stoichiometry of nucleoporins obtained in the proteomic studies, however,
indicate that the NPC size is only 44 MDa in S. cerevisiae and ~ 60 MDa in rat.
The discrepancy supports the conclusion that the NPC is a porous, lattice-like
assembly, rather than a solid entity (Brohawn et al., 2008; Hinshaw et al., 1992),
which accounts for the overestimate of mass based on volumetric analysis.
Nucleoporin
Metazoan
homolog
Nup192
Nup188
Nic96
Nup205
Nupl88
Nup93
Nup157/170
Nupl55
Domain Architecture
Abundance
Scytoplasmic
Mass per NPC
Fraction of
1Ma structured
total NPC
enucleoplasmc = 1 MDaunstrueured
0 0
*
p9
*
ine00
Nup35
Nup133
0
Nup120
Nup160 -
0
6 0
Nup107
Nup85
Nup96
Seh1
Sec13
Sec13
Mip1
Mlp2
Nup2
Nupi
Nup6O
0
0
*
*
0
0
Nup37
Nup43
Tpr
Tpr
Nup5O (I
35%
mrmm
0000
Nup133
Nup85
Nup145C
Seh1
m
e
Nup53/59
Nup84
m
a
m
0
20%
-
*
*
U
0
U
n
0
N Em
l I IIlI
12%
11ID
m
-
Nup153
Pom152
0
Ndcl
Pom34
a
0
Ndcl
9%
e
-CD-gp210
Porn121
Nsp1
Nup49
Nup62 Qe31ee 11m11m
Nup58 10
0
e
Nup54 gilDILIIIENup57
Nup159 Nup214/CAN C
Gle1
Gle1
m0
Nup42 NLP1/hCG1
7
0
m
Nup88 -(00
Nup82
Nup358/RanBP2
ALADIN
Rae1/Gle2
Nup145N
NuplOO
Nup116
Rael/Gle2
10%
man
*
ejj
Z
I
Nup98 (111JIIIIZ
Nup98
Nup98 4111
**
0
11111111 :
a
(ACE1)
0
Calculated Composition of the NPC from S.cerevissa
Cow
e-coil CD
ans domain
W aP
7
m
11.3
EE
A-foowtotal
6.5
6.3
5.3
I[D
2.0 1.31.1
5.9
6.6
46 MDa
OMan
(C ) tranhnrnrane
M FG-repeat
-Mop
Figure 1.3 - Inventory of the NPC
Summary of the nucleoporins that make up the NPC. Domain architecture of
nucleoporins from S.cerevisiae as determined by x-ray crystallography or
prediction (where structural information is still lacking). Abundance and derived
mass calculations are based on published Nup/NPC stoichiometries (Cronshaw
et al., 2002; Rout et al., 2000). Nucleoporins specific to metazoa are italicized.
Subcomplexes
The majority of nucleoporins are organized in discrete subcomplexes each
present in multiple copies that arrange according to the symmetry elements of
the NPC to form the complete structure. The subcomplexes are biochemically
defined and reflect the stable interaction of subsets of nucleoporins. Interestingly,
these subcomplexes are also found as entities in mitotic extracts of higher
eukaryotes, when the nuclear envelope breaks down during open mitosis
(Matsuoka et al., 1999). At the end of mitosis, NPCs reassemble from these
subcomplexes in a defined order (Dultz et al., 2008). Each of the eight scaffold
segments arranged around the central rotational axis is composed of 5
subcomplexes (Figure 1.2). Nup82/Nupl59/Nspl form a subcomplex localized at
the cytoplasmic side of the NPC (Belgareh et al., 1998). A second pool of Nspl
complexes with Nup57 and Nup49 and resides in the center of the NPC, forming
the bulk of the central transport barrier (Grandi et al., 1993). The scaffold ring is
constructed from two major subcomplexes: the heptameric Y- or Nup84-complex
and the heteromeric Nic96 complex. The Y complex is the best-characterized
subcomplex of the NPC and is essential for its assembly, as shown in several
organisms (Boehmer et al., 2003; Fabre and Hurt, 1997; Galy et al., 2003; Harel
et al., 2003; Walther et al., 2003). It has 7 universally conserved components Nup84, Nup85, Nup120, Nupl33, Nup145C, Secl3 and Seh1 - that assemble
stoichiometrically and exhibit the eponymous Y-shape in electron micrographs
(Kampmann and Blobel, 2009; Lutzmann et al., 2002; Siniossoglou et al., 2000).
In many eukaryotes, notably excluding S. cerevisiae, three additional proteins,
Nup37, Nup43, and ELYS/MEL-28, are considered members of the Y complex,
but their architectural role is unclear (Cronshaw et al., 2002; Franz et al., 2007;
Rasala et al., 2006). Inmost models, the Y complex is thought to symmetrically
localize to the cytoplasmic and the nucleoplasmic face of the NPC sandwiching
the Nic96 complex. The Nic96 complex is not as well defined as the Y complex,
likely reflecting the fact that it associates less stably. However, Nic96 interacts
directly with Nup53/59 (Hawryluk-Gara et al., 2005), and co-immunoprecipitation
with Nupl88 (Nehrbass et al., 1996) and with Nupl92 have been reported
(Kosova et al., 1999). Further, the Nic96 complex is the tether to the Nspl
complex in the center of the NPC. The newest defined subcomplex contains the
transmembrane Nup Ndcl, considered to be an anchor for the NPC in the pore
membrane. This complex contains Nup1 57/170 and Nup53/59, which connect
the Ndcl complex to the Nic96 complex (Makio et al., 2009; Onischenko et al.,
2009). The other two transmembrane nups, Pom34 and Pom152, are reported to
interact with Ndcl as well, albeit less strongly. Mlp1/2 are attached to the NPC
ring via Nup60 (Feuerbach et al., 2002) and likely form the nuclear basket
structure (Strambio-de-Castillia et al., 1999).
Dynamics
An important aspect of the NPC is that it is not a rigidly assembled machine, but
a rather dynamic entity. Inverse fluorescence recovery after photobleaching
experiments using GFP-tagged nucleoporins showed that different parts of the
NPC have drastically different residence times (Rabut et al., 2004). Some mobile
components detach from the NPC within seconds, while other components are
stable throughout the entire cell cycle. Notably, the components of the structural
scaffold - the Y complex and the Nic96 complex - are stably attached, while FGnucleoporins are more dynamic. These studies on nucleoporin dynamics are
consistent with the very slow protein turnover of scaffold nucleoporins (D'Angelo
et al., 2009; Daigle et al., 2001). The scaffold structure of the NPC can be viewed
as a docking site for more mobile nucleoporins, which often have functional roles
at sites away from the NPC (Kalverda and Fornerod, 2007; Xylourgidis and
Fornerod, 2009).
Domain architecture
Until about five years ago, very little high-resolution structural information on
nucleoporins was available. This was largely due to the technical difficulties of
obtaining nucleoporins of sufficient quantity and quality for structural studies, a
challenge particularly severe in the case of scaffold nucleoporins. Despite the
scarcity of experimental evidence, structural predictions grouped nucleoporins
into a small set of fold classes (Berke et al., 2004; Devos et al., 2004; Devos et
al., 2006; Schwartz, 2005). First, FG-domains, the primary transport factor
interaction sites, are present in about one third of all nucleoporins. Second,
coiled-coil domains are present in a number of nucleoporins. Third, scaffold
nucleoporins are largely composed of p-propellers, a-helical domains, or a
tandem combination of both. Using this simple classification, about 76 % of the
mass of the yeast NPC was accounted for.
FG-repeats
A total of 13 % of the NPC mass is made up of FG-repeat containing peptide
stretches. The repeats are found in terminal extensions of ~10 nucleoporins and
make up the physical transport barrier. NTRs specifically interact with the FGregions, which allow them to enter the central transport channel. How FG-repeat
regions exactly form the transport barrier is vigorously investigated and hotly
debated (Frey and Gorlich, 2007; Lim et al., 2007; Peters, 2009; Rout et al.,
2003). Systematic deletion of FG-regions from different nups has shown that the
total mass of these filaments is more important than any one individual FGfilament, arguing for substantial redundancy in the meshwork (Terry and Wente,
2007). The intrinsic disorder of the FG-filaments is well documented in a series of
crystal structures (Bayliss et al., 2000; Fribourg et al., 2001; Grant et al., 2003;
Liu and Stewart, 2005). Only short peptide stretches are orderly bound to the
convex outer surface of the HEAT-repeats that build NTRs, with the
phenylalanine sidechains inserting between neighboring helices. Otherwise, the
filaments remain without structure. Little is known about the intervening, non-FG
sequences. They are poorly conserved, but are rich in polar and charged
residues, probably important for the biophysical properties of the transport
barrier.
Coiled-coils
Coiled coils in the NPC fulfill structural roles. The nuclear basket of the NPC is
mainly constructed from the large coiled-coil proteins Mlp-1/2 in yeast and Tpr in
vertebrates. Coiled-coils are often used for protein-protein interactions, thus the
nuclear basket may serve as a general recruitment platform to bring accessory
factors close to the NPC. The desumoylating enzyme Ulpi, for example, is stably
associated with the nuclear basket (Li and Hochstrasser, 2000). Lining the
central NPC channel are 6 nucleoporins containing coiled-coil regions. The FGNup Nspl is part of two distinct entities, the Nspl*Nup57*Nup49 complex
(Grandi et al., 1993) and the Nspl*Nup82*Nupl59 complex (Bailer et al., 2001).
In both, the proteins are held together by coiled-coil interactions (Bailer et al.,
2001) and the Nspl-Nup57-Nup49 complex is, in addition, tethered to the NPC
scaffold via the N-terminal coiled-coil region of Nic96 (Grandi et al., 1995). So far,
only a homodimerized 10 kDa fragment of Nup58 (the vertebrate ortholog to
Nup57) has been structurally characterized (Melcak et al., 2007). Biochemical
analysis suggests that the network involves specific rather than promiscuous
interactions, arguing for a specific tethering function for the coiled-coil segments.
It will be interesting to see these coiled-coil interactions in atomic detail in order
to manipulate them and potentially swap the attached FG-domains within the
NPC. Such experiments could provide important insight into the organization of
the FG-network, if there is such.
p-propellers
A large portion of the NPC scaffold is build from p-propellers, one of the most
abundant classes of proteins, especially in eukaryotes, and with diverse functions
(Chaudhuri et al., 2008; Paoli, 2001). A set of Nups were initially identified as Ppropellers based on sequence analysis. In yeast, only Secl3 and Seh1 contain
the signature WD-40 repeat motif and were among the very first p-propellers to
be recognized (Pryer et al., 1993). More nups have since been recognized as ppropellers despite the lack of signature sequence motifs. The N-terminal domain
of Nup133 was the first experimentally determined p-propeller of the NPC and
after this structure was solved, the additional non-canonical p-propeller domains
in the NPC were identified (Berke et al., 2004). To date, 5 of the 8 universally
conserved p-propellers in the NPC are structurally characterized (Figure 1.4). In
Nup133, Nup120, and Nup159 (hNup214) the p-propellers are N-terminal and
seven-bladed. While forming a distinct entity in Nup133 and Nup159 (Weirich et
al., 2004), physically tethered but otherwise not interacting strongly with the C-
terminal part of the protein, the p-propeller in Nup120 is fully integrated with an
adjacent helical domain to build one continuous oblong domain (Leksa et al.,
2009). Seh1 and Sec13 are so far unique variations of p-propellers in that they
are open and 6-bladed (Brohawn et al., 2008; Debler et al., 2008; Fath et al.,
2007; Hsia et al., 2007). Their partner proteins insert a seventh blade into the ppropeller to complete the domain in trans. The function of the p-propellers is
architectural and it is widely assumed that they serve as protein-protein
interaction sites. Peripheral p-propellers can recruit accessory proteins, like the
mRNA export factor Dbp5 (von Moeller et al., 2009), whereas those more
centrally located likely are used to connect subcomplexes.
NIc96
PDB Code: 20X5, 2RFO
1
839
186
Nup8-Sehl
3EWE, 3F3F
Nup85 1
hNup133 11
349
yNup145C-hSec13
Nup120
3BG1
3HXR
3 111
1317
741
hSec13
hNupl07 1
744
Seh1 1
yNup 145
hNup1O7.hNup133
314R, 3CQC
1
517
925
111502
979
1 156
hNup133
1XKS
1037
11587S7
dhi
Nup170
315P, 3150
Nup159 (hNup214)
IXIP, 201T
1
67 - 514
387
11460
1 K322
316
rNup58
2OSZ
mNup35
1WWH
1C10
325
1l=585
327-411
920
677
10E1325
156-261
585
327-411
rNup153-ZnF-RanGDP
3CH5, 3GJ3-8
rNup153 11
974-1012
IMP8 1IEK7861
1
156-261
11Z1
Nsp1-FGs-Importin j3
2BPT
Nsp1
974 1 076
1
hNup98
1K06, 205X/Y
r~an
1
691-909
1W1216
502
hNup358-RanBD-RanGDP
1RRP
hNup358 1
hRan
1
/03224
216
Figure 1.4 - Structures of nucleoporins
Comprehensive list of all representative nucleoporin structures published to date.
PDB accession codes are indicated. Structures are gradient-colored red- or blueto-white from N to C terminus. Residue information for each crystallized fragment
is given below the structure. Structures are shown in the assembly state that is
supported by crystallographic and biochemical evidence. Structures are from
S.cerevisiae unless noted otherwise (h, human; m,mouse; r, rat). 2QX5(Jeudy
and Schwartz, 2007); 2RFO(Schrader et al., 2008b); 3EWE(Brohawn et al.,
2008); 3F3F(Debler et al., 2008); 3CQC(Boehmer et al., 2008) 314R, 315P,
315Q(Whittle and Schwartz, 2009), 3BG1(Hsia et al., 2007); 3HXR(Leksa et al.,
2009); 1XKS(Berke et al., 2004); 1XIP(Weirich et al., 2004); 20IT(Napetschnig et
al., 2007); 20SZ(Melcak et al., 2007); 1WWH(Handa et al., 2006); 1K06(Hodel
et al., 2002); 2Q5XN(Sun and Guo, 2008); 2BPT(Liu and Stewart, 2005);
3CH5(Schrader et al., 2008a); 3GJ3-8(Partridge and Schwartz, 2009);
1RRP(Vetter et al., 1999).
a-helical domains
a-helical domains make up more than half of the mass of the NPC scaffold.
Structural prediction classified the non-coiled-coil a-helical domains into a
strongly related group of a-helical solenoids (Devos et al., 2006). a-solenoids are
characterized by a two or three helix unit that is repeatedly stacked to form an
elongated, often superhelical domain with N and C terminus at opposite ends of
the molecule (Kobe and Kajava, 2000). Such regular, a-helical repeat structures
are, often in combination with P-propellers, common scaffolds in large protein
assemblies such as the clathrin vesicle coat (Edeling et al., 2006), the protein
phosphatase 2A holoenzyme (Xu et al., 2006) and the anaphase promoting
complex (Herzog et al., 2009), to name a few. Surprisingly, structural
characterization of a-helical domain containing Nups has revealed three different
a-helical folds, each distinct from a regular a-solenoid arrangement (Boehmer et
al., 2008; Jeudy and Schwartz, 2007; Leksa et al., 2009; Schrader et al., 2008b;
Whittle and Schwartz, 2009). Nic96 was the first experimentally determined ahelical structure of a scaffold nucleoporin and it showed an unexpected, atypical
a-helical topology (Jeudy and Schwartz, 2007; Schrader et al., 2008b). The 30
helices of the -65 kDa domain, excluding the -200 residue N-terminal coiled-coil
domain, are arranged in a J-like topology, forming an oblong domain. The chain
starts in the middle of the elongated domain, zigzags up on one side of the
molecule, folds back over a stretch of 7 helices and then continues past the N
terminus to the other end of the molecule (Figures 1.4, 1.5). Three other a-helical
scaffold nucleoporins (Nup84, Nup85 and Nup145C) have since been structurally
characterized and shown to adopt the same fold as Nic96, pointing to a common
ancestor (Brohawn et al., 2008) (see below). A second, distinct a-helical fold has
been identified in structures of Nup133 and Nup170, which are more distantly
related, but share an extended and stretched a-helical stack (Boehmer et al.,
2008; Whittle and Schwartz, 2009), substantially different from the first group.
The third was revealed in the structure of Nup120, which forms a domain that
fully integrates a p-propeller with an a-helical domain (Leksa et al., 2009). The ahelical segment is built around a central stalk of two long helices wrapped with 9
additional helices in an unprecedented fashion. In summary, the a-helical
domains that occur in the NPC fall in different classes that provide a significant
challenge for structure prediction methods. One obvious challenge is the
exceedingly low sequence conservation, even between orthologs, apparent in
the inconsistent nucleoporin nomenclature. Poor sequence conservation is likely
due to some degree of malleability of the scaffold structure and the construction
from common sequence elements (Aravind et al., 2006). Whether poor sequence
conservation is further the result of adaptive evolution, linking several
architectural nucleoporins to speciation, is an intriguing possibility that should be
explored in more detail (Presgraves et al., 2003; Tang and Presgraves, 2009).
A
ACE1
N!cA
NiW6
205-228
234-243
248-263
270-294
NUp14SC
188-196
217-223
229-242
253-287
NupOS
103-117
134-164
170-188
201-212
333-343290-301 244-253
20.
345-356
38-373
388-396
406-416
426-430
432-442
457-469
478-485
486-498
500-513
534-543
550-562
566-583
586-611
617-634
637-647
650-667
685-697
708-728
732-742
765-790
800-820
828-835
304-314
317-326
331-346
352-363
373-379
382-392
399-410
417-427
431-441
447-460
469-485
488-497
501-514
526-530
534-553
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d
257-266
280-292
300-317
326-339
343-348
354-362
372-381
390-398
406-411
415-427
462-475
480-491
497-509
517-528
532-543
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Nup84
n.d.
35-54
60-84
114-129
Sec31
417-424
428-435
441-455
459-462
171-190
510-519
192-203
206-218
240-253
258-268
273-277
282-305
324-335
342-352
354-368
386-404
409-422
n.d.
n.d.
n.d.
n.d.
667- 680
686-700
705-723
739-768
783-820
841-867
871-882
895-913
524-531
536-543
549-560
588-577
582-586
594-803
808-624
628-638
642-660
666-685
697-712
717-724
731-744
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Figure 1.5 - The Ancestral Coatomer Element ACE1
Four scaffold nucleoporins, Nic96, Nup85, Nup84, and Nup145C share a distinct
65 kDa domain also found in the COPIl protein Sec3l, manifesting common
evolutionary ancestry between the two membrane coats. (Brohawn et al., 2008;
Devos et al., 2004). (A) The structures of an "average" ACE1 protein (left) and
Nic96 (right) are shown in cartoon form and colored from blue (N-terminus) to
white (C-terminus). The regions corresponding to the crown, trunk, and tail
modules are indicated. The "average" ACE1 protein was made for illustrative
purposes by superimposing the known ACE1 protein structures crown, trunk, and
tail modules separately and constructing each of the 28 helices and connecting
loops in the most frequently observed position. The orientation of the modules
with respect to one another is shown in the average ACE1 case to most clearly
show the architecture and connectivity of the fold. The position of the crown and
tail modules in Nic96 are rotated 60* and 20* respectively compared to the
average ACE1 structure as indicated. (B) The amino acid assignment for each of
the canonical 28 ACE1 helices is indicated for all structurally characterized ACE1
proteins. All numbering is from S. cerevisiae except for helices 21-28 from
Nup84, which correspond the human homolog Nup107. Helices are colored
according to the module to which they belong as are the labels in (A). n.d. (not
determined) indicates helices that fall within regions of the proteins for which no
structural information is available.
ACEI domains
As mentioned above, the four a-helical scaffold nucleoporins Nic96, Nup145C,
Nup85, and Nup84 are constructed around a common -65 kDa domain
composed of 28 helices. Notably, this domain has to date only been identified
outside the NPC in Sec3l, one of the main building blocks of the COPII vesicle
coat. The commonality was surprising. Sequence conservation between the five
members is so low that no specific structural relationship was inferred previously
(Alber et al., 2007a; Hsia et al., 2007). This domain, which we termed Ancestral
Coatomer Element 1 (ACE1), is a structural manifestation of the likely common
origin of the NPC and the COPII vesicle coat (Devos et al., 2004). ACE1 is
constructed from three modules, crown, trunk and tail, that together form an
elongated molecule of ~ 140A x 45A x 45A. Structural superposition of ACE1
proteins shows that individual modules are closely aligned, while differences in
linkers between modules results in significant differences in their relative
orientations. These differences, as well as proteolytic susceptibility data, suggest
at least modestly flexible hinges connect the modules, especially the trunk and
the tail. This likely explains why all crystal constructs except for Nic96 contain
either the trunk and crown (Brohawn et al., 2008; Debler et al., 2008; Hsia et al.,
2007) or the tail (Boehmer et al., 2008). Even with the structural information in
hand, it is difficult to find additional ACE1 proteins. Beyond a few residues
conserved between orthologs, ACE1 is not characterized by a distinct sequence
motif. The reason for this amazing degeneracy on the sequence level likely is
that for folding the ACE1 domain only some general sequence profiles need to
be satisfied. For example, helices a5, a7, al5 and a17 are typically hydrophobic,
because they are incased by surrounding helices and are largely buried and
solvent inaccessible. Thus, a combination of sequence profile evaluation, ahelical prediction, and overall length are currently the only indicators for the
ACE1 domain. The two remaining a-helical scaffold nucleoporins without any
crystallographic structural information are Nup188 and Nup192. Whether or not
they belong to the ACE1 class, remains to be determined, but it appears unlikely.
Assembly
Structures of large protein assemblies are typically solved by a combination of
cryo-electron microscopy and x-ray crystallography (Chiu et al., 2006), exploiting
the strengths of both methods. With EM-data in the 10-15 A range, fitting of
crystal structures can often be performed fairly reliably, providing in effect a
composite structure at atomic resolution. In the case of the NPC, the best
tomographic EM data for the full assembly does not extend beyond ~ 58 A (Beck
et al., 2007) and thus, unfortunately, does not lend itself to directly fitting the
available crystal structures. A promising step toward closing the resolution gap is
the recent EM reconstruction of the Y complex at ~ 35 A resolution, which allows
at least tentative fitting of crystal structures (Kampmann and Blobel, 2009). Apart
from the resolution gap, however, the biggest difficulty currently is the absence of
strong experimental data on how the subcomplexes, notably the Y complex,
assemble to form the core scaffold structure of the NPC.
In an alternative approach to the hybrid method of combining experimental EMand crystallographic data, Alber et al. used a plethora of volumetric and
stoichiometric data, combined with distance restraints obtained from a
comprehensive co-immunoprecipitation analysis of all nucleoporins, to solve the
subcomplex assembly puzzle (Alber et al., 2007a; Alber et al., 2007b)). Even
though each datapoint has very limited information content by itself, a useful
three-dimensional model is generated by combining all the data, conceptually
similar to the way an NMR structure is computed. The resulting draft model at an
estimated 5 nm resolution provides a plausible arrangement of Nups in the yeast
NPC, information that is not available from the current tomographic studies.
According to the computed model, 8 Y complexes each self-assemble into a
cytoplasmic and a nucleoplasmic ring, defining the Z-dimension of the scaffold at
~38 nm (Figure 1.6, middle row labeled "computational model"). Sandwiched
between the two rings are two 8-membered rings of Nupl57/170, Nupl88, and
Nup1 92. All remaining nucleoporins, except for the filamentous nuclear basket
and cytoplasmic extensions, have been positioned as well and decorate the main
scaffold. Clearly, this model has severe limitations and will have to be adjusted
with more detailed information becoming available. For instance, the Y complex
is modeled as a compact rod in the computational model and the coat is spacefilling with very small gaps, both in apparent contrast to results from
crystallographic analyses. The exciting possibility of this combinatorial approach
however is that it should be possible to integrate currently available and
forthcoming high-resolution structures to further refine the model. To date, crystal
structures accounting for 23% of the mass of the NPC scaffold are available.
Integration of these data could potentially reveal an NPC structure that would
come fairly close to the reality.
Organization of
Y-complex
Y-complex
arrangement inthe NPC
Relationship to other
subcomplexes
TransmembraneNups
Coat Nups
ChannelNups
polo
Nup4
Nup133
Nup8s
E
o
polo
Sec
13
Nup120
se
Nupl45C
:LIL2L14L&1JOMI
4_4,04
Figure 1.6 - Assembly models for the NPC
Three recently proposed models for the structural organization of the NPC are
illustrated. The fence pole model (top row), computationally generated model
(middle row), and lattice model (bottom row) are compared in their prediction of
protein interactions within one Y complex (left column), Y complex organization
within the NPC (middle column), and placement of Y complexes relative to other
NPC subcomplexes (right column). Dashed lines to the left or underneath panels
represent -40 nm (The approximate height of a Y complex) and are shown for
scale. In the fence pole model, heterooctameric poles of Nupl45C-Secl3 and
Nup85-Sehl observed in crystal structures organize four rings of 8 Y complexes
each. These four rings form a cylinder adjacent to the transmembrane Nups on
the membrane side and layers of adaptor Nups followed by channel Nups
towards the transport channel (Hsia et al., 2007; Debler et al., 2008). The
computationally generated model provides localization volumes for each Nup and
shows 8 Y complexes arranged into two separate rings, one towards the
nucleocytoplasmic and the other the cytoplasmic side of the NPC. The other
Nups are arranged into membrane rings, inner rings, linkers between rings, and
FG nucleoporins (Alber et al., 2007b). The lattice model is based on structural
homology of ACE1 proteins in the NPC and COPII vesicle coat (Brohawn et al.,
2008). ACE1 proteins are colored by module with tails green, trunks orange, and
crowns blue. A model of a single Y complex incorporates the demonstrated
specific interactions between domains of the 7 proteins. 8 Y complexes are
assumed to form a nucleoplasmic and cytoplasmic ring, which may or may not be
connected by additional lattice elements such as Nic96 and Nup1 57/170 in an
inner ring. The illustration is meant to emphasize the predicted open, lattice-like
organization of the NPC structural scaffold and is not meant to imply specific
interactions between complexes. While it seems likely that the NPC lattice will be
composed of ACE1-containing edge elements and vertex elements made from ppropeller interactions as observed in the COPIl vesicle coat, the exact nature of
the vertex elements in the NPC lattice remains to be seen (Brohawn et al., 2008).
Blobel and coworkers have taken a different experimental approach to address
the subcomplex assembly problem. Their rationale is that the subcomplexes
assemble only at very high protein concentrations as they are found in the
assembled NPC in the living cell. Such conditions are difficult to reproduce in
vitro, but they can potentially be mimicked in protein crystals where the protein
concentration is similarly very high. Thus, crystal-packing interactions between
dimeric subcomplex fragments have been used to develop an assembly model
for the NPC (Debler et al., 2008; Hsia et al., 2007). Besides the overall
dimensions of the scaffold, the resulting model is drastically different from the
computational model. Four instead of two 8-membered rings of Y complexes are
stacked on top of each other to form a continuous membrane-proximal shell. The
rings are connected by alternating heterooctameric poles of Nupl45C*Secl3
and Nup85-Sehl (Figure 1.6, top row labeled "fence pole model"). The Nic96
complex is postulated to form a second, inner shell within the NPC that connects
to the FG-network. Similar to the computational model, the Y complexes are
envisioned to directly contact the pore membrane via membrane-inserting ALPSmotif containing amphipathic helices predicted to be present in several Y
complex components (Drin et al., 2007). However, experimental evidence
supporting membrane-insertion is so far only available for an ALPS-helix found in
the vertebrate Nupl33 p-propeller, and the prediction of ALPS-helices is nontrivial. Indeed, several predicted ALPS-helices have been found to be well
packed and to contribute to the hydrophobic core of nucleoporins (for example in
Nup85 and Nup120), making their involvement in membrane-insertion unlikely.
Comparison to vesicle coats
In a groundbreaking paper, it was postulated that the NPC and vesicle coats
share a common ancestor, dating back more than one billion years to the very
early eukaryote (Devos et al., 2004). The principal hypothesis was that these
assemblies are used to fulfill similar roles specific to eukaryotes, namely
stabilizing the highly curved membranes of vesicles and the circular openings in
the nuclear envelope. Judged by primary sequence analysis, however, the
relationship between the components is largely undetectable. One important
pillar of the 'proto-coatomer' hypothesis is that Sec1 3 is a bona fide component
of both the COPIl vesicle coat and the NPC. Structural evidence supporting the
hypothesis was provided by showing that a class of four scaffold nucleoporins
shares a specific 65 kDa domain, ACE1, with Sec3l of the COPII vesicle coat
(Brohawn et al., 2008; Brohawn and Schwartz, 2009a). The COPII coat is
organized into a membrane-proximal inner layer built from Sec23-Sec24
heterodimers and Sari, and an outer coat assembled from Secl3-Sec3l
heterodimers (Stagg et al., 2008). Sec3l in the COPII coat and Nup145C in the
NPC interact very similarly with Sec1 3 by insertion of a 7th blade to complete the
p-propeller. This interaction mode is recapitulated in Nup85 binding to the Sec13
homolog Seh1, which is also facilitated by insertion-completion of the p-propeller.
Since Nup145C, Nup85, and Sec3l all share the same ACE1 domain, the
structural data not only suggests a common ancestor, but it also provides clues
as to how the NPC assembles.
Incomparison to the NPC, the COPII vesicle coat is much simpler, has been
extensively studied, and its assembly is quite well understood. Most informative
with respect to the NPC assembly is the organization of the Sec1 3-Sec3l outer
coat of COPII. Here, two Secl3-Sec31 heterodimers dock via their ACE1 crown
modules to form an edge element (Fath et al., 2007). Four edge elements
converge in a vertex, where the N-terminal p-propellers of Sec3l interact. COPII
vesicles of different sizes can be assembled from different numbers of edge
elements by adjusting the angles at the interaction sites (Stagg et al., 2008). In
analogy to the Sec31*Sec31 homodimer interface in the COPIl coat, it was
predicted on a biochemical and structural basis that Nup145C and Nup84 form a
heterodimeric edge element via a crown-to-crown interface in the NPC. Due to
the predicted steric conflicts, this arrangement would be fundamentally
incompatible with the hetero-octameric fence pole model discussed above (for
details, see (Brohawn et al., 2008)). We predict that the entire NPC scaffold has
an open, lattice-like organization with significant similarity to the COPIl coat, also
built from vertex and edge elements (Figure 1.6 bottom row labeled "lattice
model", Figure 1.7). As much as COPII and clathrin lattices seem not to share
common construction principles (Fath et al., 2007), so do similarities between
NPC and clathrin lattices not extend beyond superficial characteristics.
Figure 1.7 - Predicted lattice-like arrangement of Y complexes in the NPC
The predicted arrangement of Y complexes (blue) in the NPC with respect to the nuclear
envelope (pale yellow) is shown in a cut-away side view on the left (with 10 of the 16 Y
complexes in the NPC shown) and from a top view on the right. The Y complexes are
predicted to be part of an exterior lattice in which ACE1 edge elements of
Nup84-Nup145C are arranged parallel to and stabilize (but not directly contact) the
positive curvature of the nuclear envelope. This is analogous to the role of ACE1 edge
elements of Sec3l in the COPII outer lattice.
One important distinction between the computational and the hetero-octameric
pole model on one hand and the lattice model on the other is that in the latter the
Y complex and likely also the Nic96 complex components Nic96 and Nup157/170
do not directly coat the membrane, although there may be punctual contacts. By
analogy to the Sec23*Sec24 inner coat of COPIl, adaptor molecules instead are
predicted to directly contact the pore membrane. The transmembrane Nups,
especially Ndcl, are prime candidates for this function. Indeed, Ndcl has already
been shown to physically interact with the Nic96 complex via Nup53/59
(Onischenko et al., 2009). Ndcl is found in all eukaryotes and is essential,
though this could also be related to its additional role in the spindle pole body
(Winey et al., 1993). Such adaptor-mediated anchoring to the pore membrane is
consistent with the membrane-proximal gaps observed in cryo-tomographic
reconstructions (Beck et al., 2007; Stoffler et al., 2003).
Obviously, there are some important limitations to the analogy to COPIl. First, it
is not yet apparent how the vertices are formed in the NPC. In COPIl, Sec3l
contains an additional N-terminal p-propeller, absent in the ACE1 domains of the
NPC, but essential for COPII vertex assembly (Fath et al., 2007). Thus, the
vertex might look different in the NPC. Further, the COPII coat is a self-enclosed
lattice structure that completely encapsulates the positively-curved membrane
vesicle. Inthe NPC, the lattice can be continuous in lateral direction around the
central transport gate, but must terminate on the nucleo- and cytoplasmic sides.
Further, the circular pore opening has positive curvature only in the axial
direction, but negative curvature in equatorial direction. Since all vesicle coats
seamlessly wrap positively curved membranes, it is not unreasonable to
speculate that a specific architectural element might occur exclusively in the NPC
to establish its unique geometrical requirements.
Outlook
Progress toward the structural characterization of the NPC at high resolution, one
of the holy grails of structural biology (Bhattacharya, 2009), has been spectacular
since our last review on the subject, a review that was intended as a roadmap
toward this ambitious goal (Schwartz, 2005). The past five years have seen
improvement in images of the entire NPC available, and a flurry of crystal
structures of various key parts of the NPC. New computational methods were
developed to integrate geometric data from various sources, and these were
applied to the NPC, in part to demonstrate a general means to tackle other large
assemblies (Alber et al., 2008). The path toward a high-resolution structure of the
NPC will continue to be highly interdisciplinary. Single-particle reconstruction on
subcomplexes has still been used only sparingly, but holds great promise to
close the gap between the resolutions provided by crystallography and by
tomography. It is likely that integration of structural data, particularly
crystallographic data, into computationally derived models will provide further
useful information. Finally, developments in super-resolution microscopy promise
to allow in vivo measurement of distances within the NPC on the order of 10 nm,
a potential source of further useful parameters - parameters so far inaccessible
experimentally. These distances would complement other available structural
data. Judging by recent progress in the field, dramatic developments in our
understanding of the NPC lie just around the corner.
Acknowledgements
We are grateful to Elena Kiseleva and Ohad Medalia for providing images for
Figure 1.1. We thank all members of the Schwartz laboratory for discussion and
suggestions. We sincerely apologize to those whose work we have referenced as
a result of unintentional oversight. T.U.S. is supported by the Pew Scholars
Program and by NIH grant GM077537.
Chapter two
Structural evidence of a common ancestry of the nuclear pore
complex and vesicle coats
This chapter was previously published as Brohawn, S.G.*, Leksa, N.C.*, Spear,
E.D., Rajashankar, K.R. & Schwartz, T.U. Structural evidence of a common
ancestry of the nuclear pore complex and vesicle coats. Science. 322, 13691373 (2008).
*These authors contributed equally to this work.
S.G.B. and N.C.L. designed, conducted and analyzed biochemical, biophysical
and crystallographic experiments and wrote the manuscript; E.D.S. designed and
conducted S. cerevisiae experiments; K.R.R. assisted with crystallographic data
collection and obtained the first SAD substructure solution; T.U.S. advised on all
aspects of the project and wrote the manuscript.
Abstract
Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These
massive assemblies comprise an eightfold symmetric scaffold of architectural
proteins and central-channel phenylalanine-glycine-repeat proteins forming the
transport barrier. We determined the nucleoporin 85 (Nup85)-Sehl structure, a
module in the heptameric Nup84- or Y complex, at 3.5 angstroms resolution.
Structural, biochemical, and genetic analyses position the Y complex in two
peripheral NPC rings. We establish a conserved tripartite element, the ancestral
coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat
proteins, providing structural evidence of coevolution from a common ancestor.
We identified interactions that define the organization of the Y complex on the
basis of comparison with vesicle coats and confirmed the sites by mutagenesis.
We propose the NPC scaffold, like vesicle coats, is composed of polygons with
vertices and edges forming a membrane-proximal lattice that provides docking
sites for additional nucleoporins.
Introduction
Exchange of macromolecules across the nuclear envelope is exclusively
mediated by NPCs (D'Angelo and Hetzer, 2008; Tran and Wente, 2006; Weis,
2003). Whereas much progress has been made understanding the soluble
factors mediating nucleocytoplasmic transport, the structure of the -40-60 MDa
NPC itself is still largely enigmatic. Cryo-electrontomography (cryo-ET) and cryoelectronmicroscopy (cryo-EM) have established the NPC structure at low
resolution (Beck et al., 2007; Drummond et al., 2006; Stoffler et al., 2003).
Crystal structures of scaffold NPC components are emerging (Berke et al., 2004;
Boehmer et al., 2008; Hsia et al., 2007; Jeudy and Schwartz, 2007), but the
resolution gap still precludes fitting into the cryo-ET structure. Overall, the NPC
has eight-fold rotational symmetry with an outer diameter of -100 nm and a core
scaffold ring ~30 nm wide. The central FG-repeat containing transport channel
measures -40 nm in diameter, defining the maximum size of substrates (Pante
and Kann, 2002).
The modularity of the NPC assembly suggests a path toward a high-resolution
structure (Schwartz, 2005). Of the -30 bona fide nucleoporins (Nups) that
comprise the NPC, only a core subset is stably attached (Rabut et al., 2004). In
Saccharomyces cerevisiae, this core includes two essential complexes: the
heptameric Y complex and the heteromeric Nic96-containing complex (hereafter
called the Nic96 complex; unless noted all proteins are from S. cerevisiae). The
Y complex is composed of one copy each of Nup84, Nup85, Nup120, Nup133,
Nup145C, Sec13 and Seh1. It self-assembles from recombinant proteins in vitro
and forms a branched Y-shaped structure (Lutzmann et al., 2002). Deletion or
depletion of individual components of the Y complex leads to severe assembly
defects in many organisms (Fabre and Hurt, 1997; Galy et al., 2003; Harel et al.,
2003). The Nic96 complex is less well characterized, but appears to contain the
architectural nucleoporins Nup157/170, Nup188, Nup192, Nup53, and Nup59
(Lusk et al., 2002; Marelli et al., 1998; Zabel et al., 1996). p-propellers and
stacked a-helical domains form the building blocks of the constituents of the
Nup84 and Nic96 complexes (Devos et al., 2006; Schwartz, 2005). Because
vesicle coats (including COPI, COPII, and clathrin) share similar elements, a
common ancestry has been hypothesized despite very low sequence homology
and the absence of experimental structural evidence (Devos et al., 2004).
A recent computer-generated model of the NPC based on a plethora of primary
data from different sources placed the Y complex at the NPC periphery,
sandwiching the Nic96 complex in the center (Alber et al., 2007b). In contrast, a
model solely based on the structure of the Nupl45C-Secl3 heterodimer and
crystal-packing interactions was proposed that is inconsistent with the computer
model (Hsia et al., 2007).
Results
We solved the structure of a complex of Nup85 residues 1-564 (of 744) and
intact Seh1 (referred to as Nup85-Sehl) at 3.5 A (Table 2.1). Seh1 and Nup85
form distinct units in a tightly associated complex (Figure 2.1). Seh1 folds into an
open six-bladed p-propeller structure. The blades fan out consecutively around a
central axis, typical for canonical p-propeller structures (Chaudhuri et al., 2008).
Between blades 1 and 6, the N-terminus of Nup85 is inserted and forms a threestranded blade that completes the Seh1 propeller in trans. Following its Nterminal insertion blade, Nup85 forms a compact cuboid structure composed of
20 helices, with two distinct modules, referred to as 'crown' and 'trunk'. Helices
al-a3 (residues 100-200) meander along one side of the trunk; the other side is
formed by helices a12-a19 (residues 362-509) running in the opposite direction
in an antiparallel zigzag to the C terminus. The trunk elements are separated by
an intervening crown composed of helices a4 to al1 (residues 201-361) that
form a distinct bundle that caps one end of the trunk. Helices a5 to al 0 in the
crown module are almost perpendicular to the helices in the trunk.
Table 2.1 - Data collection and refinement statistics
Nup85 1.se4 -Sehl
Native
Nup85-Sehl
Selenomethionine
Data collection
Space group
Wavelength (A)
Cell dimensions
a=b, c (A)
P41212
0.9792
P41212
0.9792
112.6, 350.5
112.5, 351.2
a=#3=y (0)
90
90
Resolution (A)
40-3.4
10.7
13.7 (1.8)
96.9 (98.8)
3.1 (3.3)
50-3.7
12.7
21.9 (1.8)
99.1 (99.9)
6.3 (6.1)
Rsym (%)
/ l
Completeness (%)
Redundancy
Refinement
Resolution (A)
No. reflections
Rwork
/ Rree
No. atoms
Protein
Water
30-3.5
28300
32.6 / 36.9
9636
0
Wilson B-factor (A2 )
119
R.m.s deviations
Bond lengths (A)
0.008
Bond angles (0)
1.2
Coordinate error (A)
0.58
Nup85-crown
C-term
Nup85-trunk
Seh1
+
900
a18
a15
C-term
Wa19
-
~
~
Figure 2.1 - Structure of the Nup85-Seh1 complex
The structure of the heterodimeric Nup85-Sehl complex is shown in two views
(A, B), related by a 900 rotation around the horizontal axis. Nup85 has a trunk
(orange, helices al-a3 and a12-a20) and a crown (blue, helices a4-al 1) module.
The p-strands at the extreme N-terminus of Nup85 form an insertion blade, which
complete the Seh1 (green) P-propeller. (C) 2Fo-Fe omit map (contoured at 1.20)
with a Ca-trace of the Nup85-Sehl complex.
In the asymmetric unit of the crystal, two heterodimers are aligned along a noncrystallographic dyad, generating patches of contacts (Figure 2.2). This
interaction is unlikely to be functionally meaningful because the contact residues
are poorly conserved in orthologs. Moreover, analysis of Nup85-Sehl by
analytical ultracentrifugation (AUC) showed a single species of -104 kDa with a
hydrodynamic radius of 4.4 nm (Figure 2.3). This hydrodynamic radius is close to
the theoretical value calculated from the atomic coordinates using HYDROPRO
(Garcia De La Torre et al., 2000) and reflects the elongated shape of the 103
kDa Nup85-Sehl complex (a spherical protein of 220 kDa would have the same
radius). Gel filtration also showed that Nup85*Sehl is a single 103 kDa
heterodimer at concentrations up to 20 mg/ml (Figure 2.4). Hence, we restrict our
analysis to this heterodimer.
B
C
Figure 2.2 - Arrangement of two Nup85-Sehl heterodimers in the
asymmetric unit
(A) Association and orientation of the two Nup85-Sehl heterodimers in the
asymmetric unit. The heterodimers form an interface of -1800 A2 and associate
lengthwise along a two-fold axis. (B) Surface conservation of Nup85-Sehl in a
view 900 rotated from the lower molecule in (A) with outlines corresponding to
contact regions involved in forming the interface. Red and blue outlines indicate
contacts made with Nup85 and Seh1, respectively, and conservation is shaded
from white (not conserved) to orange (conserved). (C) The electrostatic surface
potential of the Nup85-Sehl heterodimer (colored from red (-8 kT/e) to blue (+8
kT/e)) with outlines as in (B). Yellow and blue outlines correspond to contacts
made with Nup85 and Seh1, respectively. The view is the same as in (B).
6.3 pM
B0A-
U
031
-
3.2 pM
--
0.6 pM
0.2.j
01
0.01
3
Sedimentation Coefficient (8)
4L
6.00
625
I
6.50
075
Radha
700
00
2
M
m
&62$
710
aMUSM
K0050.20
00
600
7.00
an0u5
0.5
0.4
0.3
0.2
0
-0.1
0.5
1
15
2
Radius^2 - Radius(ref)^2 In an
0.03
0.02
8
0.01
0
-001
-0.02
05
1
15
2
RadIus^2 - RadIus(ref)A2 In cm
2.5
Figure 2.3 - Nup85-Sehl is a dimer in solution as determined by analytical
ultracentrifugation
(A) C(s) distribution analysis of Nup85-Sehl sedimentation velocity data.
Sedimentation data from three concentrations of Nup85-Sehl were analyzed
globally in Sedphat (Schuck, 2000) with a hybrid local continuous distribution and
global discrete species model. Data was fitted from 2 to 10 s-13 with fixed partial
specific volume. Residual plots for each concentration are shown below. The
48
sedimentation coefficient and corresponding rmsd values for the samples in
order of decreasing concentration were 5.56 S13, 5.58 S13, and 5.60 S-13 and
0.0079, 0.0056, and 0.0045, respectively, which corresponds to a single species
with a molecular weight of -104 kDa and frictional ratio (fIfO) of 1.43. The
calculated molecular weight for Nup85-Sehl is 103 kDa. (B) Sedimentation
equilibrium analysis of Nup85-Sehl. Sedimentation data from 6 concentrations at
three speeds were analyzed globally in Ultrascan 9.0. The data was best fit by an
ideal single species model. The top panel shows data points as yellow triangles
with fit curves overlaid (13.5 krpm - dark blue, 17.5 krpm - medium blue, 22.8
krpm - light blue). The lower panel shows residuals of the fitted curves. The
molecular weight was determined to be 99 kDa with a standard deviation of 0.4
kDa, closely matching the results obtained via sedimentation velocity.
2500
200020 mg/ml
C1500a-
M1000
10 mng/m
-15 mg/ml
-- 1mg/ml
----- Nup145C-Sec13
8
10
12
14
Elution Volume (mL)
16
18
Figure 2.4 - Nup85*Seh1 is a dimer in solution as determined by size
exclusion chromatography
Elution profiles of the Nup85 1.564-Sehl complex at 1, 5, 10, and 20 mg/ml show a
single peak eluting at 12.2 ml on a Superdex 200 10/300 (GE Healthcare)
column indicating a hydrodynamic radius of 4.4 nm. The hydrodynamic radius
was independently determined by sedimentation velocity and is consistent with
the value calculated from the experimental crystal structure using HYDROPRO
(Garcia De La Torre et al., 2000). The elution profile of Nupl45C-Secl3 is shown
for comparison (dashed black line).
The connectivity and topology of secondary structure elements and the threedimensional folds of Nup85*Sehl and Nupl45C*Secl3 (Hsia et al., 2007) are
remarkably similar (Figure 2.5A), despite very low sequence identity between
Nup85 and Nup145C (10%) and moderate identity between Seh1 and Sec13
(32%). Like Nup85, Nup145C has an N-terminal three-stranded P-sheet that
provides a seventh blade to close the open p-propeller of Sec1 3. The trunk and
crown modules of Nup145C are also similar to those in Nup85, although their
relative orientation is modestly different in the two proteins.
Nup85-Sehl
Nupl45C-Secl3
C
176
176
-__
-.
-160.-
Nup145C-SecI
Nup$4+C
Nup145C1
S13
15g, --
u8
Nup145C4UEA4ec13
Nup4+Wp14SC4EA.Sec3
125,
125
100
10
75,
C
10
12
14
5
1$
lUtin
Voume(at)
10
12
14
18
ElufonVokm*("A)
300
25M --
-
-- Nup84wil
NUp145Catalf-Set
3
250 -
'"-NupB4Atal+NUP14CtH-5ec13
NUpSOtUI-DICD
N.P1ASCAa2.Sec13
NupS4al-D5CD+Nu4p145Cata.5eCt3
~150
200.
10
0
14
10
12
Volume("q
EMIOn
8
S
10
12
14
Is
Elutn Voham ("l
90*
Nup85-crown
Time (mn)
Nup1 45C-crown
20
60
40
80
100
120
0.00
0
CD
-
).02
-
.04
:x
Nup4 + Nup145CAta-Sec13
Nup84 + Nup145CataI-EllEA-Sc13
NuS4Atail-DSICD+ Nup145CAtaf-Sec13
-1
0
-20
-
-30
-
0
-40-
8as
-5
54ereviae
DLLPYLSDTCAVSFDAVS SIELtKQYPKD
C9J1w494SCLAKYLKENCETTSTIFE
&Jjrus:SQLLOIC9---TVCVFVT
A004p+JI ELL SY LAOKCSATIITMVQ
DAhisoM ISY-EELEENAPELF$VIQ
c.aIm*,olU Y-QELKEKCPEt.YAVIG
YxpaIyvoSCA-CEVO#SCQETLEQfl
FIOLVKNYPLTIIIESYPL4
VN LLOGYPSFSMLLKNYT5M
OTLLSN1'TSY
ILSRYPQGPC-
c SVIZSytC NOPRIRDLA
cgubta~AAKIALD~0SNP,,IS)145M.C NOPRLKALA
KJaws SKTAlIK
K SVL TLLC NDPIVEIA
A905SyuiiIT-rTA$ASN
AVtYTL.C NDPLVRELS
D.Ilansen+
,INLA)M05N A
YS'IILTLID$NUDAVKSTA
Ca7sicanSIETAIQTN 514SVVITL5D NDVVVRSIA
YKhpWoiyc4CAArLKTN VI4CVVJ 5MMC 5.ASAQSTA
M
g
N =1.02* 0.04
Ko=S*2nM
AH= -55*3kJimol
T
=432kJJ/Mo
=
g
U
W
Scerevouol~aSKLAIEK
0.0
0.5
1.0
Molar Ratio
1.5
.
Figure 2.5 - Comparison of Nup85-Sehl and Nup145C-Sec13 and
identification of the Nup84-Nup145C crown-crown binding interface
(A) The topologies of the Nup85-Sehl (left) and Nup145C-Sec13 (right, PDB
accession code 3bgl (Hsia et al., 2007)) complexes are shown, illustrating an
overall similarity with three shared structural elements - trunk, crown, and ppropeller. Colors are assigned as in Figure 2.1. (B) Surface representations of
the crowns of Nup85 and Nup145C are shown colored according to electrostatic
surface potentials (top) and sequence conservation (bottom) in a view rotated
90* from (A). Sequence conservation is based on the phylogenetic tree of
budding yeasts (Dujon, 2006) and is colored from white (not conserved) to
orange (conserved). A partial sequence alignment of helix a8 (indicated by
arrows in (A)) is also shown with surface exposed residues indicated by green
dots, residues buried in the hydrophobic core by blue dots, and residues not
modeled in the structure by dashes. Mutations made in this helix in Nup145C are
shown above the sequence alignment and the corresponding residues are
outlined in the surface representations of Nup145C. (C)In the upper panels, gel
filtration data of Nup84 alone, Nupl45C-Secl3 (wild type or -ELIEA mutant)
alone, and Nup84 plus Nup145C-Sec13 (wild type or -ELIEA mutant) are shown.
The shift in the Nup84 plus wild type Nupl45C-Secl3 chromatogram indicates
complex formation and is absent in the case of the -ELIEA mutant. In the lower
panels gel filtration data of Nupl45C-Secl3 alone, Nup84 alone (wild type or DSICD mutant) alone, and Nupl45C-Secl3 plus Nup84 (wild type or -DSICD
mutant) are shown. The shift in the Nup145C-Sec13 plus wild type Nup84
chromatogram indicates complex formation and is absent in the case of the DSICD mutant. (D)Isothermal titration calorimetry data illustrating high-affinity
binding for wild type Nupl45C-Secl3 and Nup84 (black). Experimental values for
N, KD, AH, and TAS are shown. In contrast, binding is lost for both crown-surface
mutants Nup84-DSICD (grey) and Nup145C-ELIEA (red).
The most conserved regions of Nup85 are involved in the interaction with Seh1.
The corresponding interface between Nupl45C and Secl3 is also well
conserved, but Nup145C has an additional highly conserved surface on the
crown module around helix a8 that is not observed in Nup85 (Figure 2.5B). This
region is reasonably polar and poorly conserved in Nup85 but highly conserved
and distinctly hydrophobic in Nup145C, suggesting a protein-protein interaction
site. Nup84 and Nup120 bind to roughly opposite sides of Nupl45C*Secl3 in the
Y complex (Lutzmann et al., 2002), and the C-terminal helical region of Nup145C
is necessary for binding Nup120 (Figure 2.6). Thus, we hypothesized that the a8
crown surface of Nup145C is the binding site for Nup84.
-
NUP&5(fl)-Sehl
Nupl45C-Secl3
NupOS(fl)-Sehl + Nupl45C-Secl 3
225
Ektbn Volume (mQ
-
Nup8S-Sehl
NupI20
Nup85-Sehl + Nupl 20
15
17
Ekstion Vokmm (nd)
-
-
EkMm VaWnu (rM
Nupl2O-Nup85(*Sehl
Nupl2O-NupSS(*Sehl+Nupl45C.Secl3
Nupl2O-Nup85(*Sehl+Nupl45(Atafl-Secl3
Nupl45C-Secl3
Figure 2.6 - Nup120 binds Nup145C and Nup85 via their tail modules
(A) The heterodimeric Nup85(fl)-Seh1 and Nupl45C-Secl3 complexes were
analyzed on a Superdex 200 HR26/60 column. A mixture of both complexes
(orange) does not result in a higher molecular weight species indicating that the
complexes do not directly interact. (B)The heterodimeric Nup85-Sehl complex
(without the Nup85 tail module) and Nup120 were analyzed on a Superose 6 HR
10/300 column. Again, a mixture of both samples (orange) does not result in a
higher molecular weight species. (C) Nup85(fl)-Sehl including the tail module
binds Nup120 (blue), and adding Nup145C-Sec13 results in a pentameric
complex (orange) (Superdex S200 HR1 0/300). This complex is not formed when
the tail module is removed from Nup145C (dashed). Taken together, this series
of experiments demonstrates that the tail modules of both Nup145C and Nup85
are responsible for Nup120 binding.
To test this hypothesis, we mutated the Nup145C sequence VLISY in a8 to
ELIEA, introducing two negative charges and eliminating a conserved aromatic
side chain on the crown surface (Figure 2.5B). The overall structure of Nup145C
did not appear to be perturbed by this modification: (i) Nupl45C-ELIEA*Secl3
bound to Nup120 to form a 1:1 complex indistinguishable from Nup145C-Sec13
in gel-filtration experiments (Figure 2.7); Nupl45C-Secl3 and Nup145CELIEA*Sec13 complexes (ii) had comparable thermostability (Figure 2.8) and (iii)
showed identical behavior in gel filtration (Figure 2.5C). The ELIEA mutation
completely eliminated Nup84 binding. In isothermal-titration calorimetry (ITC)
experiments, Nup84 bound wild-type Nup145C-Sec13 tightly (KD = 3 ± 2 nM; 1:1
stoichiometry) but not Nupl45C-ELIEA-Secl3 (Figure 2.5D). Similarly, Nup84
formed a stable complex with Nupl45C-Secl3 but not with Nup145CELIEA*Sec13 in gel filtration (Figure 2.5C). We conclude that the Nup84 binding
site on Nup145C includes the exposed surface of helix a8.
-
Nup12O-Nup85(fl)-Sehl + Nup145C-ELIEA-Sec13
Nupl20-Nup85(fl).Seh1 + Nupl45C(Q691G).Secl3
125
150
175
200
225
250
275
Elution Volume (ml)
Fractions
7-19
*-NupI2O
"N=lS-E1EA.Sec13
I-. uO5f)
I-Seh I
Fractions
9-18
Fractions24-26
+-Nup120
NuplSC(Q0691G)-Secl3
SNupS
4--Sehl
Figure 2.7 - A single point mutation in the predicted interaction helix of the
Nup145C tail module disrupts Nup120 binding
Formation of a pentameric Nup120-Nup85(fl)-Seh1-Nup145C-ELIEA-Secl3
complex (orange) is disrupted by the Q691G mutation in the tail module of
Nup145C (dashed) (Superdex S200 HR26/60). Formation of the
Nupl20-Nup85-Sehl complex is unaffected by this mutation. Fractions were
analyzed by SDS-PAGE.
-o- Nup145CAta8-S.c13
-- Nup146CAtaI-ELEA-Sec13
-20-
-26-
20
30
40
60
60
70
80
Temperature (*C)
0-
-10-
.1.
20
-
30
4
60
Nup84Atan
Nup84AtaI-DSICD
70
Temperature ( 0C)
Figure 2.8 - Surface mutation of the Nup145C and Nup84 crowns does not
negatively affect protein stability
(A) Heat denaturation of Nup1 45CAtaiI-Secl 3 and Nup1 45CAtaiI-ELIEA-Secl 3
monitored by circular dichroism. Ellipticity at 208nm is plotted as a function of
temperature. The melting temperature, Tm, is nearly identical at ~52 C for both
protein complexes. (B) Nup84Atail and Nup84Atail-DISCD also have very similar
thermal denaturation characteristics (Tm, -41 "C)indicative of uncompromised
protein stability.
To determine the consequences of abolishing the Nup84 binding site on
Nup145C in vivo, we introduced the Nup145C-ELIEA mutation into the NUP145
gene in yeast. Strains carrying NUP145-ELIEA in a ANUP145INUP84-GFP or
ANUP145INUP133-GFP background displayed a marked defect in incorporating
Nup84-GFP and Nupl33-GFP into the NPC (Figure 2.9, 2.10). Compared with
wild type, a significantly larger fraction of GFP-tagged protein was found in the
cytoplasm, indicating that the Nup84 binding interface on Nup145C is crucial in
recruiting both Nupl33 and Nup84 to the NPC (Figure 2.11). In addition, nuclear
pores were clustered into discrete foci on the nuclear envelope of the strains
expressing Nup145C-ELIEA, indicative of severe NPC assembly defects and
similar to Nup84 and Nupl33 null strains (Doye et al., 1994; Siniossoglou et al.,
1996). Cells expressing wild-type Nup145C demonstrated the expected punctate
nuclear rim staining in both Nup84-GFP and Nupl33-GFP strains. Thus,
disruption of the Nup84 binding site on Nup145C affects NPC assembly and
function and causes loss of Nup84 and Nup133 from pores. The loss of Nup133
can be rationalized because it is attached to the Y complex through a binary
interaction with Nup84 (Boehmer et al., 2008; Lutzmann et al., 2002). Some
Nup84 and Nup133 proteins remain associated with nuclear pores in the
Nup145C-ELIEA expressing strains, arguing for the existence of additional
weaker attachment sites for both proteins in the NPC. It has been shown that an
ALPS membrane-binding motif is present in Nupl33 (Drin et al., 2007). Because
Nupl33 and Nup84 are tightly associated (Boehmer et al., 2008), the ALPS motif
might be weakly functional in recruiting Nupl33*Nup84 to the NPC even when
the Nup84-Nup145C interaction is compromised.
DIC
Nup133-GFP
DNA
Overlay
DIC
Nup84-GFP
DNA
Overlay
NUP145
nup145C-ELIEA
NUP145
nup145C-ELIEA
Figure 2.9 - Elimination of the Nup84 binding site on Nup145C results in
nuclear pore assembly defects in vivo
(A) NUP145/NUP133-GFP and NUP145-ELIEA/NUP133-GFP or (B)
NUP145/NUP84-GFP and NUP145-ELIEA/NUP84-GFP were grown at 24 *C
and visualized by fluorescence microscopy. DIC, GFP-fluorescence, DNA
(visualized with Hoechst dye), and false-colored overlay (GFP fluorescence green, DNA - blue) images of the same field are shown in columns from left to
right.
vector
nup145A
NUP133-GFP
NUP145
nup145C-ELIEA
vector
nup1458
NUP84-GFP
NUP145
nupl45C-ELIEA
SMM-leu
SMM-leu
+ 5-FOA
NNIh
B
/I
240C
300C
370C
Figure 2.10 - Growth analysis of yeast strains
(A) The nup145C-ELIEA mutant supports viability. nup145A/NUP133-GFP and a
nup145A/NUP84-GFP strain carrying SBYp1 15 (NUP145/CEN/URA3) and empty
vector, or vector encoding NUP145, or vector encoding nupl45C-ELIEA were
grown in SMM-leu overnight, serially diluted and grown on SMM-leu or SMM-leu
+ 5-FOA plates at 300C for two days. (B) nup145C-ELIEA is lethal at elevated
temperatures in rich media. A nup145A strain carrying plasmid-borne NUP145
(SBYp116) or nupl45C-ELIEA (SBYp117) were grown in minimal medium at
24*C. Serial-diluted cells were plated onto YPD plates and grown for 2 days at
240C, 300C or 37*C.
A
NUP145
T
S
nup145c-EL1EA
P
T
S
P
TSP
Nup1 33-GFP
TSP
Pgk1p
(cytosol)
nup145c-E LEA
NUP145
T
Nup84-GFP
S
qflow
P
TSPo
T
S
P
HZ- -- I
Pgk1p
(cytosol)
Figure 2.11 - Nup133-GFP and Nup84-GFP become more soluble in the
Nup145-ELIEA strain
(A) NUP145 NUP133-GFP (YS221) and nupl45C-ELIEA NUP133-GFP (YS222)
or (B) NUP145 NUP84-GFP (YS223) and nupl45C-ELIEA NUP84-GFP (YS224)
strains were spheroplasted, and total lysates (T) were separated into 16,000xg
soluble (S) and pellet (P)fractions. Equal cell equivalents from each fraction
were analyzed by immunoblotting using rabbit anti-GFP or monoclonal anti-Pgkl
antibodies.
On the basis of lattice packing observed in crystals of Nupl45C-Secl3, Hsia et
al. (Hsia et al., 2007) proposed that Nupl45C-Secl3 and Nup85*Sehl each form
heterooctameric poles that span the entire NPC in a "concentric cylinder" model
of NPC structure. However, the Nupl45C-Secl3 lattice contacts involved in the
putative heterooctamer overlap with the crown surface of Nupl45C shown here
to be the Nup84-binding site. Additionally, Nupl45C-Secl3 and Nup85-Sehl
behave nearly identically during gel filtration, indicative of heterodimers when
their large hydrodynamic radii are taken into account (Figures 2.4 & 2.5). AUC
experiments confirmed that Nup85-Sehl is a heterodimer in solution (Figure 2.3).
Thus, the heterooctameric pole model (Hsia et al., 2007) is inconsistent with our
results.
The structural similarity between Nup85 and Nupl45C extends to at least three
other proteins (Figures 2.12 & 2.13). First, the architectural nucleoporin Nic96
(Jeudy and Schwartz, 2007) shares a common structural core (Figure 2.13) but
has a distinct N terminus (Figure 2.12). The shared cores mutually superimpose
with an rmsd of 3.0-3.5 A. Nic96 has a trunk module (al to a3 and a12 to a19), a
crown module (a4 to al1), and an N-terminal coiled-coil extension (instead of the
insertion blade of Nupl45C and Nup85) that tethers it to the FG-containing Nspl
complex (Grandi et al., 1995). Apart from the N-terminal differences the three
proteins differ mainly in the relative orientation of the crown and trunk modules.
Although a previous comparison of Nupl45C to the COPIl coat component
Sec3l did not reveal a strong similarity (Hsia et al., 2007), comparison with
Nup85, Nupl45C and Nic96 shows that Sec3l has corresponding trunk (al to a3
and a12 to a18) and crown (a4 to al1) modules. Sec3l homodimerizes to create
an "edge element" in the COPII coat by an internal domain swap between two
crown modules (Fath et al., 2007). This domain swap results in a mixed crown
module that is identical in topology to the unmixed crowns in Nup85, Nupl45C,
and Nic96 (Fath et al., 2007). Structural prediction using Phyre (Bennett-Lovsey
et al., 2008) also places Nup84 in the group containing Nic96, Nup85, Nupl45C,
and Sec3l. Similarity extends beyond the trunk and crown modules to a 'tail'
module that has been structurally characterized in the C-terminal domain of
human Nup107 (homolog of yNup84) and in Nic96 (Boehmer et al., 2008; Jeudy
and Schwartz, 2007) (Figure 2.13C). The last three helices in the tail module of
Nupl07 form the interaction site with Nupl33 (Boehmer et al., 2008). In Nic96
this region is predicted to be a protein binding site as well (Jeudy and Schwartz,
2007). Because we find this characteristic tripartite structural element of crown,
trunk, and tail in architectural proteins of the NPC and the COPIl coat, we term it
the ancestral coatomer element 1 (ACE1).
NIc96
Nup85
Nup145C
Nup84
Sec31
crown
trunk
2
tail
crown
trunk
tail
N-elaboration
N-elaboration
N-elaboration
N-elaboration
Figure 2.12 - Architecture of ACEI
(A) ACE1 containing proteins are shown as cylinders and sheets. Crowns are
shown in blue, trunks in orange, tails in green, and other domains in gray.
Modules with predicted structures are shown half-transparent. (PDB accession
codes 3bgl, Nup145C; 2qx5, Nic96; 3cqc, Nup107; 2pm6, Sec3l) (B) Cartoon
illustrating the similarity and modular nature of the ACE1 element. The N-terminal
elaborations are for Nic96 a coiled-coil domain that interacts with the Nspl
complex, for Nup85 the Seh1-interacting insertion blade, for Nup145C the
Sec13-interacting insertion blade preceded by an autocatalytic cleavage domain
and Nupl45N, and for Sec3l the Sec1 3-interacting insertion blade is preceded
by its own N-terminal 7-bladed p-propeller. Sec3l has a unique proline rich
insertion C-terminal to its trunk module followed by a conserved region predicted
to be a-helical. The structure of this region was difficult to predict with high
confidence. Thus, it remains to be determined whether Sec3l has a tail module
similar to those in Nic96/Nup1 07/Nup85/Nupl 45C.
~~
*Nup85
-7
010
*ul5
09
a12~
a2
Nupl45C
W4*NUP85O
C
pa
5
Figure 2.13 - Superposition ACE1 modules
Superposition of (A)crowns, (B)trunks, and (C)tails of ACE1 with known
structures (PDB accession codes 3BG1, Nup145C; 2QX5, Nic96; 3CQC,
Nup1 07; 2PM6, Sec31). Helices are labeled according to Nup85 in (A)and (B)
and Nic96 in (C). In (A) Helix a4 of Sec31 and the short p-sheet between helices
a5 and a6 in Nic96 are omitted for clarity. The rmsd between modules is 2.9-3.2A
in (A), 3.4-3.5A in (B)and 2.7A in (C).
Can we predict ACE1 functional sites from established interactions? Analogy to
Sec3l monomers in the COPII edge element suggests that Nup145C and Nup84
might interact crown to crown. Based on the Phyre-model and structural
alignment, we constructed a surface point mutant replacing two conserved
hydrophobic residues on helix a8 of the Nup84 crown with aspartate (Nup84ISICM to Nup84-DSICD) (Figures 2.8 & 2.14). Nup84-DSICD disrupts Nup145C
binding in a manner analogous to Nup145C-ELIEA, severing Nup84 binding as
shown by gel filtration and ITC (Figure 2.5C,D) Thus, the Nup84-Nupl45C
interface is a crown-crown interaction involving a8 helices as in Sec3l
homodimerization. Additionally, we found that the tail modules of Nup145C and
Nup85 are necessary for interaction with Nup120 in a manner analogous to the
human Nupl07 interaction site for human Nupl33 (Boehmer et al., 2008)
(Figures 2.6 & 2.7).
A
B
C
a9'
aT
a8
M540
M535
2068
8323
8324
M537'
S21
Np84
09
010
Sec3l
Nupl45C
Nup84
Figure 2.14 - Surface mutations of the Nup145C and Nup84 crowns
The crown helix a8 is highlighted in (A) Sec3l (PDB code 2PM6) (B) Nup145C
(PDB code 3BG1) and (C)Nup84. In (A), one Sec3l monomer is shown half
transparent and the three hydrophobic residues buried in the interaction between
crown helix a8 in each monomer are shown as sticks. In (B), the three exposed
a8 residues in Nup145C mutated to disrupt binding to Nup84 are shown (V_Ll|Y
to ELIEA). In (C), the two exposed hydrophobic a8 residues in the Phyrepredicted Nup84 structure mutated to disrupt Nup145C binding are shown
(ISICM to DSICD).
Here we have shown that ACE1 is abundant in the two main scaffolding
subcomplexes of the NPC. To date, Nic96 is the only ACE1 protein in which all
three modules (crown, trunk and tail) are structurally defined. In Nic96, the three
modules form a continuous, rigid hydrophobic core (Jeudy and Schwartz, 2007;
Schrader et al., 2008b). In the other four experimental structures, only a subset
of the modules are present. We speculate that the three modules within ACE1
can allow hinge movements, utilized to different extents in specific family
members.
Discussion
ACE1 is different from regular a-helical repeat structures, including HEATrepeats and TPR-repeats (as discussed in (Jeudy and Schwartz, 2007)). The ahelical modules that compose ACE1 are distinctly irregular, most notably with
elements that fold back onto themselves forming a U-turn within the crown
module. The trunk is composed of two zigzagging helical units running in
opposite directions. We propose that this architecture confers rigidity to the trunk
and thus distinguishes it from regular helical repeat structures that are often
inherently flexible (Conti et al., 2006). As a consequence of the specific
arrangement of the helices in ACE1, several helices in the trunk and crown are
encased by neighboring helices and thus have a characteristic hydrophobic
character (typically helices a6 and a1O, Figure 2.15). This pattern of hydrophobic
helices may help to find additional ACE1 proteins. Several sequence elements,
notably in the crown and at the predicted hinge regions, distinguish ACE1 from
other a-helical domains (Jeudy and Schwartz, 2007). Nevertheless, these
characteristics are subtle enough to remain undetected in typical primary
sequence (i.e. BLAST) searches and candidate proteins need to be examined
using all available tools, including phylogeny and secondary and tertiary structure
analysis.
A
Nup85
ltereisa
Cogabrata
KJacus
Agossy
Dthmnsonu
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YJapo4tla
MTID-----DSNRLLMOVFDFLDGTAQLSNNKTDEEEQLYKRDPVSCAILVPM
51
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MANDEFA--DTQDLLMDV NLDF
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DF
MGDQEPS- -TLSQKFDDI
LEIP
--MPEYT--NFESKFDOI
LEIP
MFSAPPATINCADNYISP PITAR
41
52
44
34
41
52
01
lcerewsaee
Cogabrat
KJAWs
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ahaen
C.aibcans
Y.14whria
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Cgabrte
Kjactis
Agossyp
Dnse.w
Cancans
YJipoptica
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FCEAEMETILDCDDT----ELPEITLPGAPI
02
-02
33
TVNDQPIEKNGDKMPL-KFKLCPLSYQNMAF---------ITAKDKYKL PVIIPR
DADKFPKESHN--SPL-VFKLGNGANENIMH---------VKSQKDVN
VPLST
----- PIEQPS-AKEL-RFKYNNVSSRSLAF---------DNSTKDNKLPVRFLH
PSTQQP--- DL--EKL-RFKFAPVLSRSFAF---------NCGSRTGTLUVQVPY
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DSSDSSKSSYDQWSPVAIFKSDKEYIDLSDWLNT---QKSLEFKFDVQK RKCIEK
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97
85
92
85
79
94
L-D-TSKE SA
S-D-FSEN IN
L-D-ESKE AQ
V-D-NSKE3S
N-D-VSKS TK
SIP-FDEK IL
G-DEWSDN KG
138
126
133
126
133
147
128
VSC
EIYRDLCDDRVF------NVPTIGVV-------NSNFAK
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KIIEGFTNDDITRIDLEDDEDPICLISTSAKFNSARKA
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SK
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Cgfabrata
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105
as
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VENLTQY
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HAN
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188
177
182
175
183
197
176
4a5
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Ypa~ka
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YHNCAACQSCFFCSQSTYEVRQP
FETQY
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SVKDSTAGKKV
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IS
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I
F P DPELLN
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NLVLKLSQAFCSSATDISGE----DY E FLL
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IF
Agossuyp
YPRE-LEPV REDLVLQLNLNWSQSENKISIE----SS EICLL
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YJipo*tica
GPGN-VSFY
NSISTVQSKLLKIN---DAK----KG TLLQ
K LQYST
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K EFSQY
KUIYYSK
Dhaseni
T
INCDK
Coaicans
T SSYCDT
YApoticA
GTNHT
I
NQR
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SKN
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344
326
330
SFCGFL
I-PSLELSA LQMSLEANV-VDITND--------W
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I-PSEELIQE GLAVSKTP-IDVTNP--------W
SYCGLM YI-PTLELSQ
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DAAVANYP-VOTFTV--------W
390
372
376
373
388
403
373
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Cogabrt
Kiactis
AgossyW
204
276
282
277
277
291
280
010
a9
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239
221
230
222
222
236
226
L
L
F
L
327
332
347
327
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a13
PCVDI
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Iceevisist
C94gla ae
Ki1actis
AgoMS)k~
Djhanenii
C.&lbcans
Y.Iipo"yfCA
a14
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VSVDTI
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ACASHA KGSLLLAETLENC
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LATAAFVALCEAKC KTDFY ----SCTAAFTAAj.CEAKGUENDLL-- - -- DDCGLE
SATAAYVARLLELKCUNNYYS -- AIN -----D-VTVT
PPTTAYLAELMELRF*RGYYF
NCY ----------- CAA
SVIAALVSNM#CYDAC
(317
0316
Icerevitae
combra
C/acmi
Aw~ssypq
ahomeniit
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thIpo~ra
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DNEMLEDLFSYRNGMASYMUNSFU FELCSLGDKE3WUPVA#
DDIEIDDIFSQRNGMASFL MNF LECSMKMKT PVAI ---- ISLSPYNTPS
---ISLSSDSGDS
SGLNSELAFENEPTICEFL ItQV LSLCSYDDKTWPVS
VSNTGDEMFCLGNTMASYL[NQF LSIVTYEDKNIWAVAI
- - -- IYLTPNYSDA
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SSASFEDLLN-RRIVSEYFITRHr DCL--NIHE VPVG 2 LLNNVVCTSNNAVS
----NPAALPDYH TNL LQCIC- -YDNIN
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ale
SIcerewlsAt
coglbrata
K.iacis
Agos"*~
aanscna
C.Albkcans
V.hrilotica
AKINE
L H F
SKKSTA EL HYPFI
SKRAI I EL HFPY1
AKIMMU EL
YPFC
N NINTES E
H*Y ECRI
ANIKVATF*NYCI
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Y
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lkiL
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kNL
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KAHNNNNLM
022
021
Slceftwvsia
Cgdbrata
k.Iacfis
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IEWR PEIAKE YTTLINQikUSAHNIIE
kKWR PDIAKE
TTL NQM5DNCNTVE
IKWR PDVAKT YKILIQDT3YQNNIVE
023
SIANSR ------------ AGKYELVKSYSS-L L EASCMECQKLDDP-VLNAIVS
AIAP4SK--------------AGQYELVKHYS -LL ENAAFQGKPMEDE-VLSAIVD
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SLMUVNCYOPOSMTNESN-ECMKQVHHII DI DDSLVNNRPIKDE-LINNIIC
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0i24
i.rvsiae
Coslbrats
Clacris
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ahanunm
C"abkcaos
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coulbrata
kactis
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D~sansenR
C.albkans
Y.ilpol
ica
KNSPAEDDVI IPQDILDCVVTNSMRQTUAU
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HKVDT------------- CFEIHPVIRQC SE
KKVD------------- LNFKIIPVQQC 1
DPAS-------------- VSDVPAEVAECH
a26
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FSNVENE- -RWSKA- --- LQ
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FRATEDC- - RWAEA- --- LK
FK SL EQ S- - RSTTAMDNL S
V ET LPQRDI K LSNK -- - LS
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----
02
--------------IE
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LLLL
EVIITKWDDAD
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KLVHIIR
NH uKKFYPinLCQII-P-FLIDSCMKFQLPDLIVIUELIoTFEFQAN
KLIH
NY KKFVP LAQFI-P-FLSEPNTTFQLPDLVIj ELLDNYESDRN
L 1 LK
FE PtTEL
IHQLINYVSPISQQFNFSTSQFMTL SAFOKWDKSQC
--
G--
---------------------
-----
£.Cerewslit
Cglabrata
Ciacris
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Y.Ipolptici
PSSMATLLKNLUKKLNFK
TDSVIAYEKE-------------------- SEE-- IA
PKELKSMEKLIKKLNGK
DSCVALYSQ*RNDD----------------- NST--CSN PESVDSFIQDVEKTLNYK
SIcerewsiat
C.94abrata
K.1acus
Agossypki
Oahansenua
C.aibkcans
Yrjgooipica
LCQA FM--LCLNLM-- I
I CQ S I'm-- -
EKSLYT--------------I1--EADKS
EITTAMYKEE-------------------- LVK- -VOT PKDPLMLINAIITKLSLK
ODEYTECES3 YKYSISNIENTTESYDWRNILQKSCNT PKDLKSLIKLL N EVVAK
EKELKECAD YLYSINNIESDADENDWRKLVC--KDN
IPKDIDSFTRLL NLIAAK
LCtHQ FM - - -
CKVYVD$M
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-
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BNupl45C
J1
s cerevislae
-b
CgIabrata
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YIlpoI).ca
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-
-
D
0
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16
LSN--NDAKNKTREIEQERRLNTKKY.-.---- NPCSFGNGHLVIGDNRSR
149
FPS-- fKKDLLTYQSVKRERRI4KMW -------.FAKFNING;QLLLKD. -PS
192
FNT--FQKDMDEFKSIRRQRRLDSAPS ------FVRFNNDSTITMKT--OK
195
IFPE--FNRDSLSMNHVSTP-- INS ---------------------KK--FS
259
4TS D- - F NKNV LKVSTP TKKK------------------------------------ 228
TNTLVNVAEEEYNKAAEK--LR.LPSYSAAVESSLAIFQPSG;QLAVKL-.PH 302
PLt
GKSGV--SIKR'LPTELQRKFLF-DOVYLDKEIEKVTI ARIK-S P
ISS S - LL
KIL--ITNQVLVKDELP-AVLTD-RTIFDNELKTTLI-HSRSS NIYCVFKKE-LI
QLSC--CKQIVFQSNLP-INKSSFONVFASYLNTSIV-NSR-P V , LVCKCS- LQ
STSCCTVTASPLPLQTQ-RSSID--SVLKKSLIDSTt-ELR-tN N- IVKQFS-LT
NLEGS--- SLEMYEEVY-PRNVS--KVYyHMLSRSMI-STR-S KIIVESNSNFS
--- LSVVDOIDESQIDIY-IDNIS--TIFHKLLSIVI-ctH-C EF KIDKTTCFE
SDL--TIGTVSKLCNFS-ENA-QO0NFLGCSVKEDMTA-Ao-RNY RYS ------
217
199
242
245
307
276
346
W
3
Scerevislae
C-9tabrta
kliactis
A.gossy~i
D~hansenY)
Calbicans
Y-Ilp
S.ceI'ewsrae
C.glabrita
K.actis
A.gMSyll
o~hansenl
FKDALOYMEKTSSDYNLWKLS
FKDVLQCtILPTSESYK. LNFS
VDNLASAYSDVPNEDK ILK1L
FDOIAAAYKPIPAEYRIWKLA
FADISLNENSTDEEEQ LKLC
FKDI ITSIIQEREE-KDV TILC
I
DPVSYPYKTD--NDQ--VKMALLKKERH
I
DPIQIDCPME----------- AKDATLKYKRR
I DPLSLPYAVNSPE --------VEKVLIKKQtH
I OPISVSKSRSHPDA -VKDVLVKKKQY
A
DEHKLNEYDEYKDVNISDSHLVKYLENLQQK
ADNLTINEP---- NPA--ISAALVDNTRK
DNICNIKDVPHVRDN~fSL
QKIi
SSTALCLSEYIGSAPVDCLQARELEASLARK
3.99
CRLTS I VSQIGP E IEEKI'R-NSSNE 'Q IFLY LLLNDVVR3S K L)(, E-,-K C sv
IDVDN
KK
I-C V STTP LEK1F L HL L IN E iE TK
I A0
51
AKLCGRIVDETRAEIDSMI S--TASO (.QKILLFLSVNDIVN
NSKTAIKKS
ELLC ItINEINSEVCAK&A- -SA CP LEK IF LYL VK RD II
TAAI A
300
345
KNFTEjLKVYNGST I EQL EKNKSDOM LEY IFI KVC C Y LK 0 INIADNA
KLLCO LKNY NSAT, .EK L IEYKNDPLETTFIYMCSGD*1K 1 I ETAITN5
Y.11POI3ycaQRLSQ CEKAVFEQVQSE ----CDNSVENIVTLLTCNRVDE' CAACLT
Calbicaos
09
S.,cerevisiae
C.glabraira
Klactls
A.gos$ypil
L YLC
LISLG,
LILLG
LVLLC
all
o~hanisenll
Lalbcans
yiigoolyrica
350
378
452
010
-NDPRIRDLAELQ KWSTCG--CStDKNISKIY
NDPRLKALAIIT
EQWSGIG--SN EIYVAKtYK
NDPIVRElAQL
TfKWKSLG--SI :DPTVISIY
NDPLVRELSTSH SKIKKLG--SSLDINIJKIYQ
o12
LS SPFECLFSLKE
LS ELFKGFFLIE
LT NPFASTAL-VN
LT SP FAESAN- SV
375
354
398
403
468
427
502
a13a1
LESEFSWLCLLNLTiC yIIUEYSLESL VQSIL---KFSLPYD---DP
ICY' FQL
NAEDVNWLVLVGAA1H .. EVDELSLEDLIGNSMAC -L--S0UICS---CVFYL ,LKL
ESNKFSRLVNLGLQY Y, DIDSLTLEDLIMNAISNPFVDTSSVSTFENLSMN'IML
ISEGLSWLATLGLQIF~l DIDALSLRELI ERGLEYSCKDQWPLN---ODI SAO LRIL
V LSC LPW ISLA IK FY D- tcrLK LH EL, IEF-QDC I----VES- --CPI Y D-1r Lt
ILETLPWNLCLALKF.F
NYNI--KKLINEF--SSSIPIC---NPVCO
LHA
KARKLOWLREFGLRLWF, RNRDISAS--IAQ-IQSS-------SS ---OWDOLR tilL
015
321
419
V
O-hanstoni
I LID NDAVKSTAVNQ
YWSDTSSLSI PKPIVKIHK LS5D--------F SE
Calbicans
VITLSD NDVVVKSIAQNQNKRTSPAVK
. D-FQP -------YllpaI)4Ica
MC S-ASAQSTAQAQIIWQSTNAMDL PDYTQ
IAANI--DVVC
5.eiese
C.9librato
K.JAczS
A.gossypi
266
245
291
296
363
322
a16
42
404
454
456
515
473
546
a17
Scerevisiae
YA-ANE.NTEKLYKEVRQRTNALDVQFC YLIQTL RFNGTRV FSK ET SDEA .TFA"AA
C.glabrata
FC-STRNAQQVLQEILKESNALGIDFL HCLQALESNGIADINNPFCoQITVQYAQ
Klactis
FACPT IPV EQL LDE LRHS EI FDVR LCFTIMLQRE--- DITEHLRDRf
LEFID
A.gassypl
tYC-SDVTPPDILVGNLKISSNNLDVRLSIFFIQILTRD---0ISPSLRDIL ,L YVE
D.JiaisenllYN-Q IHTKDK.N(ALQLI KSSHLN IK LK3FFNKVLS-RGDASF-EILSTDL SSFN
C.albkans
YV-N&IOLIE---SVTSSSLNIKLK LFCKVLA-O-FN--- YDTI 'KEF(D
"JPolyflca
YA-GKYNLEQTL--TGKAFDT, V LCAF F- LQGHAK EAPDTADR LT LQYAA
480
459
507
508
568
514
595
SxcereWslae
Q "EFAQLIICHSLF sc-FfND KAAEDTIKR ,,MRE TL1,RAST---ND-IIILNRLK
Cglabrata
Ng3ELSCHIPEALY fICAH KO) HLAKKLFES 'FSN5HHLTLD---G;KPLAIMSKLM
K.Iactis Q KLDRMIIKEALFVACF AD AIAKNTIOL I:,SSE3LYFSN--- DIK-PI:LERLQ
Agossypii
Q3KLNRMFCEALFIMCF IND RLAKQQ[VD H LS 5Q3T F FS QD -- -SNYELTR
0-banseni
F3EKIGLWXESIFVYSW SD KEN ERVI R Nt 15N3DQ IK S P-D E E -Y ITK VL K
Calbicans
y1 SS IDYWK ESTV FA HTN NDTGDAITK INSK1 IS IIKSLT IDK EQ- YA.1EVLK
YJpo4JaSMQVNALIFS
SAYAQA ITKV AR
IARKLD -------LRSYR
532
512
m5
56
622
56
643
Q21
020
Iceisiae
Cqgkbrata
K deals
A40esyO~
cthameopl
cankdans
YUpabtkac
*PSQLI FNAQ ILKDRYEGNYLS3VON3 LCSSYDLU EMAIVTS
RL LUSNNPVQ
YlUCt4D- VPNEV IYRS4 IQAKYRKCHQEE LEY L KSKDTRL KEVFITK I
KLI3NAKW-N
ISAP I IIR4LI ELYEKYSCDHLSE SN KACDFKE ELVTITT
IPKSSYYAFLI ELLDKYNRNHLS RN KACHFQEU EKVVtVS KLUDCSA- VPQS LI YEAV *IQEHSLCNYWIEE EATAKLWKK HECIIKEL LTUSND-tPRMV IYKAV1
IHPQLLSESE
LLARYENRPV
VRYAESL
LDAELWCAK1NTTVLNDV
I VE~TS
A KGDA-QKNDFJ
Q-
022
C123
a24
Ictwsat
Coata
Kiactis
A.9OSSybI
Dahamnoi
C4lbic~ns
Y.Ilpo4?ic
NNELKT REI N4E PDSER--DKWSVSINVFEVYLKLVLDNVETQ ------- ETIDS
-SKLLS
S EQ ORNS---- -TQRNDLKV#DYVIQFK-KNSENS------- NI KS
NDYLSIVETL
R PSH--TIP1WEKCLGVYEKIKLSLHNSLDP------- NI IHQ
Iceretwsie
cgebra
Kikais
A.go$Sppi
t~hansenmi
Caltlcahs
Yispipcsc
LISCNKIFYDQYKHCREVAACCNVECVSKIUEKNNP--SICOSKAK&LE-LPE
LASELPQFYSKHQMFENVTACCNIl UNNYLSAVUDLDND-SKTKEFSNLVCN-MPE
LINTLPNLCKDYSHHELVNVASSRI EAVSELFIINLNM-LSRKIPKESLLS-LPU
LVRVLPVLATDFCSHRELSVVCCV KLCHII ENYRQALETPSFKDRLLA-LP3
LLSNLPLLQ-DYNTSK-CRIASKLqKKVCDIA NYAD-QI--GNIKGLILS-L PI
LL~PTISH-KVL~IFCNIEDF--NTRKL-L
GVSHMSKAL EEKSQF E-TRVALHV
DDIQRSL LAPTTDD- -CTKAL EVLNEYT
lcevsiae
Coglabrata
K.Ijc~s
A.gossyem
D.hfinsedvi
C.Amkd ans
Y.Iipotkai~
GQPIEKAYLRC- EFAQDLMKCTYK I
T
GOP ERKYATL- HFCSR ------GQPESNYLKK-VLSS--------- L
GQPETIYLKR-ALAS--------- T
--- N- I
GEVERNYFN I-RLQLVGDVNKR FFE L-RLAES-------- K
SMKQCVYSACTRFKAS--F-S
-ANLQT RQL ET PAQ- -QMETWTHCLVEKYLQIALDNNHNQ------- EL SD
-ESKN R
SI. AK PESCHIIPLWSQCAGIVDNFVSLSQEEIQEKASLDVHTLUVS
-PEEKS
NV FK PENGLIISDWNKGACIYCKYL-IVLQNESDL ------- SAKF
-QS-- EDYV
PSPERHIPTWKECCKVVLDYARFKLSKAVDV------- LEAN
@125
C
Nup85
Nup145C
MTIDDSNRLLMDVDQFDFLDDGTAQLSNNKTDEEEQLYKRDPVSGAILVPMTVNDQ56
-------------------- DLLFSECNDEIDNAKLIMKERRFTASYTF------- 151
01
a2
Nup85
Nup145C
03
at
PIEKNDKMPLKFKTGPLSYQNMAFITAKDKYKLYPVR
- - - - K SG
-M
LK I -G
SV
IR
02
pi
PRLDTSKEFS--AYVS
P E
RK LD
Y D
110
192
02
03
02
Nup85
Nup145C
LFEIYRDLGDDRVFNVPTIGVVNSNFA--- KEHNATVNLAMEAILNELEVFIGRLK 163
EIEKVTIEA------------------RKSNPYPQISESSLL--FKDAL-DYME--- 226
03
03
03
Nup85
Nup145C
04
DQDGRVNRFYELEESLTVLNCLRTMYFILNGQDVEEN------- RSEF I ESLLNWI 212
------ KTSSDYNLWKLSSILFD--- PVSYPYKTDNDQVKMALLKKERHCRLTSWI 273
a4
as
a5
Nup85
Nupl45C
07
G6
NRSDGEP--DEEYIEQVFSVKDSTACKKVFETQYFWKLVNQLVLRCLLSQAIGCI 265
V-SQICPE IEEKIRNSS- ---------------- NEIEQIFLYLLLNDVVRASKLAI 312
uS
07
08
Nup85
Nupl45C
09
ERSDLLPYLSDTCAVSFDAVSDSI ELLKQYPKDSSSAFRE-WKNLVLKLSQAFGSS 320
ESK- ------------ NGHLSVLISYIG--------- SNDPRIRDLAELQLQKW--- 344
08
09
010
Nup85
Nup145C
011
ATDISGELRDY- IEDFLLVIG-CNQR---KILQY--SRTWYESFCGFLLYYI--PS 367
-- STGGCSI DKNI SK IYKLLSGSPFEGLFSLKELESEFSWLCLLNLTLCYGQIDEY 397
a10
012
Nup85
Nup145C
012
al
014
013
LE--LSAEYLQMSLEANVVDITNDWEQPCVDI ISG-KIKHS
-------ILPVMESLD 413
SLESLVQSHLDKFS---- LPYDDPIGVIFQLYAANENTEKLYKEVRQRTNALD-- 447
a13
015
a14
015
Nup85
Nup145C
016
SCTAAFTAMICEAKCLIENIFEGEKNSDDYSNEDNEMLEDLFSYRNGMASYMLNSF 469
VQFCWY L IQT LR FNG-------------------------- TRVF SK ETSDEATFAF 478
a16
o17
a17
Nup85
Nup145C
a18
019
AFELCSLGDKELWPVAfGLIALSATGTRSAKKMVIAELLPHY--PFVT
NDDIEW 521
AAQLEFAQ---LHGHSLFVSCF--LNDDKAAEDTIKRLVMREITLLRASTNDHIlN 529
018
a19
020
021
a22
Nup85
MISICVEWRLPEIAKEIYTTLGNQMLSAHNIIESLANFSRAGKYELVKSYSWLLFE 577
Nup145C
RLKIPS-QLIFNAQALKDRYEGNYL- -------------- SEVQNLLLGSSYDLAEMA 571
020
a21
an
Nup85
Nup145C
an
a25
ASCMEGQKLDDPVLNAIVSKNSPAEDDVI IPQDILDCVV-TNSMRQTLAPYAVLSQ
IVTSLCPRL-------LLSNNPVQNNELKTLREILNEFPDSERDKWSVSINVFEVY
an
an
Nup85
Nup145C
FYEL-RDREDWCQALRLLLLLIEFPYLPKHYLVLLVAKFLYPIFLLDDKKLMDEDS
LKLVLDNVETQETIDSLISCMKIFYDQYKHCREVAACC ---------- NVMSQEIV
024
an
Nup85
Nup145C
632
620
687
666
C25
an
WsO
VATVIEVIETKWDDADEKSSNLYETIIEADKS-LPSSMATLLKNLRKKLNFKLCQAFM 744
SKILEKNNPSICDSKAK--LLELPLCQPEKAYLRCEFAQDLMKCTYKI - - - - - - - - - -
712
an
Figure 2.15 - Sequence alignments
Multiple sequence alignments of (A) Nup85 and (B) Nup145C covering the
phylogenetic spectrum of budding yeasts. The alignment for Nup145C begins
after the N-terminal unstructured region at residue 123. Sequence alignments
were performed with 3D-Coffee (O'Sullivan et al., 2004) (using the known
structures) and illustrated with Jalview (Clamp et al., 2004). Sequence
conservation is colored from white (not conserved) to orange (highly conserved).
The secondary structure is shown above the sequences and was assigned using
information from the known structures and predictions from PredictProtein (Rost
et al., 2004). Dashed lines denote the C-terminal regions absent from the known
structures. (C) Pairwise alignment of Nup85 and Nup145C from S. cerevisiae.
The alignment was made by combining DALI (Holm and Sander, 1995) results
using the structures with an alignment from T-Coffee (Notredame et al., 2000)
corresponding to the tail modules. The secondary structure for Nup85 and
Nup145C is shown above and below the sequences, respectively, and was
assigned from the structures and predictions from PredictProtein.
Based on distance constraints and stoichiometric considerations, the heptameric
Y complex has been placed in two concentric eight-membered rings on the
nucleoplasmic and cytoplasmic faces of the NPC (Alber et al., 2007b). But how is
it oriented, and how is it connected to the inner ring of the scaffold? Nup1 33 is
anchored to the structural scaffold by its interaction with Nup84, positioning it at
the periphery of the pore (Boehmer et al., 2008). Nup84 is the link between
Nup133 and Nup145C. Thus, we position the extended arm of the Y composed
of Nup145C-Sec13, Nup84, and Nup133 facing outward (Figure 2.16). Excluding
the Nup1 33-Nup84 pair, the remaining pentamer forms a roughly symmetrical
triskelion that conceptually resembles the vertex elements that form polygonal
cages in vesicle coats. EM analysis showed that the triskelion measures
approximately 20 nm between the tips (Lutzmann et al., 2002). An eightmembered ring of the Y complex around the central transport channel has a ~50
nm diameter if the edges were to touch at the tips. Alternatively, the Y complexes
might connect through a yet unidentified adaptor protein.
A
Seel13
Sec13
B
inner ring
Figure 2.16 - Current model for the Y complex and the structural scaffold of
the nuclear pore complex
The ACE1 proteins Nup85, Nup145C, Nup84, Sec3l, and Nic96 are colored
according to Figure 2.12. (A) Schematic diagram of COPIl outer coat
organization. The Sec3l -Sec1 3 cuboctahedron composed of 24 edge elements
(Sec3l -Sec1 3 heterotetramers) is shown unwrapped and laid flat in 2
dimensions. The Sec3l -Sec3l crown-crown interactions make edge elements
while propeller-propeller interactions are vertex elements (Fath et al., 2007). (B)
Schematic diagram of the predicted lattice-like organization of the structural
scaffold of the NPC. The entire scaffold (8 scaffold segments) is illustrated
unwrapped and laid flat in two dimensions. The Y complex comprises the nuclear
and cytoplasmic rings, while the Nic96-containing complex makes up the inner
ring. The relative position and interactions between the seven proteins in the Y
complex are shown with Sec13, Seh1, Nup133, and Nup120 colored in gray. The
remainder of the complex (Nup1 57/170, Nup1 88, and Nup1 92) is illustrated in
gray. The illustration is not meant to predict relative positions of proteins or
structure of the inner ring per se, rather it is meant to show the lattice-like
organization of the structural scaffold similar to vesicle coating complexes.
Two of the three interface types observed in the outer COPI coat are also found
in the NPC coat (Figure 2.16). Nup145C and Nup84 heterodimerize via their
crown modules similar to Sec3l homodimerization, and the insertion of a seventh
blade into an incomplete propeller domain is a recurring theme in Sec1 3-Sec3l,
Sec13*Nup145C, and Sehl*Nup85. Because Nic96 shares an ACE1 element,
we predict that the inner scaffold ring is branched and lattice-like, as are the
peripheral rings. We postulate that the Y and Nic96 complexes are both vertex
elements in the assembly of the NPC structural scaffold. This would generate a
lattice-like NPC coat similar to clathrin and COP coats (Fath et al., 2007; Fotin et
al., 2004) (Figure 2.16B). This model explains how the relatively small mass of
Nup subcomplexes fills the large volume observed for the scaffold structure of
the NPC (Alber et al., 2007b) and is generally consistent with low-resolution
images of NPCs (Beck et al., 2007; Stoffler et al., 2003). Notably, COP and
clathrin cages do not directly contact membranes, but use adaptor protein (AP)
complexes to span the ~10 nm gap between the surfaces (Owen et al., 2004).
Consistent with a related architecture, a similar sized gap has been observed
between the scaffold ring and membrane surface in intact NPCs (Beck et al.,
2007).
The modular nature of COP and clathrin coats enables the construction of
assemblies varying in composition and size (Cheng et al., 2007; Stagg et al.,
2008) because the polygonal elements can be arranged in different ways. If the
same principle applies to the NPC, it could explain the existence of a subset of
NPCs that do not obey eightfold rotational symmetry (Hinshaw and Milligan,
2003) or further allow for the assembly of NPCs of distinct composition, possibly
tailored to more specific tasks. These possibilities are now testable and will bring
us closer to fully understanding the many functions of the NPC (See Appendix for
additional discussion published separately).
Methods
Protein production and purification
Full-length Seh1 and a proteolytically-mapped Nup85 fragment (residues 1-564)
from S. cerevisiae were cloned into the bi-cistronic pCOLA-Duet bacterial
expression plasmid (EMD Biosciences) using the BamHl/Notl and Ndel/Xhol
restriction sites, respectively, resulting in N-terminally His-tagged Seh1 and
untagged Nup85 and transformed into E. coi BL21(DE3)-RIL. Nup85 1.564-Seh1 is
referred to as Nup85-Sehl in the text for simplicity. Cells were grown at 30 *C in
Luria-Bertani broth supplemented with 0.4% glucose to OD600= 0.8 and induced
with 0.2 mM IPTG at 180C for 18 hours. Cells were harvested by centrifugation,
resuspended in 40 mM potassium phosphate pH 8.0, 500 mM NaCI, 20 mM
imidazole, and 3 mM p-mercaptoethanol, and lysed using a French press. The
crude lysate was centrifuged at 15,000g for 15 minutes. The soluble fraction was
then incubated with 1 ml Ni-NTA per 1000 ODs for 30 minutes at 40C and loaded
onto a disposable column (Pierce). The column was washed with four bed
volumes of 50 mM potassium phosphate pH 8.0, 400 mM NaCl, 30 mM
imidazole, and 3 mM P-mercaptoethanol and the Nup85-Sehl complex eluted in
4 bed volumes of 50 mM potassium phosphate pH 8.0, 250 mM NaCl, 250 mM
imidazole, and 3 mM -mercaptoethanol. Eluted protein was dialyzed against 10
mM Tris/HCl pH 8.0, 150 mM NaCI, 0.1 mM EDTA, and 1 mM DTT and the
6xHis-tag cleaved with PreScission protease. The protein was purified by anion
exchange chromatography on a HiTrapQ column (GE Healthcare) via a linear
NaCI gradient and twice by size exclusion chromatography using a Superdex
S200 26/60 column (GE Healthcare) run in 10 mM Tris/HCI pH 8.0, 150 mM
NaCl, 0.1 mM EDTA, and 1 mM DTT.
Selenomethionine derivatized Nup85-Sehl was prepared by growing cells in M9
Medium (Sigma) supplemented with 1 mM MgSO 4, 6.6 [tM CaC 2, 1 ml FeSO 4
4.2 g/L, 0.4% glucose, and 100 [d 0.5% (w/v) thiamine at 370C (modified from
(Doublie, 1997)). At OD600= 0.5 solid amino acid supplements were added (100
mg/ml L-lysine, L-phenylalanine, and L-threonine; 50 mg/ml L-isoleucine, Lleucine, L-valine, and L-selenomethionine). After 30 minutes, cultures were
transferred to 22*C for 20 minutes and then induced with 0.2 mM IPTG for 18
hours. The selenomethionine-derivatized Nup85-Sehl complex was purified as
described for the native version. Incorporation of selenomethionine was
confirmed by mass spectrometry (data not shown). Both the native protein and
selenomethionine derivative were concentrated to 30 mg/ml for crystallization.
Full length Nup85 in complex with Seh1 (Nup851- 744-Sehl, referred to as
Nup85(fl)-Sehl) was cloned and purified identically to Nup85 1-564 *Seh1.
Full-length Sec13 and Nup145C1 09- 712 from S. cerevisiae were cloned into the bicistronic pET-Duet bacterial expression plasmid using the BamHI/Notl and
Ndel/Xhol restriction sites, respectively, resulting in N-terminally His-tagged
Secl3 and untagged Nupl45C. In order to stabilize the complex, the C-terminus
of Secl3 and N-terminus of Nup145C were linked with a short flexible linker to
generate a single chain. This linked version behaved identically to the complex
made from separate chains with the advantage of increased stability and is here
referred to as Nupl45C*Secl3 for simplicity. The C-terminal tail module of
Nup145C was removed from the construct to generate Nup145C1 09-555*Secl 3 by
PCR and is referred to as Nup145CAtail in the text. Nup145C-ELIEA-Sec13 and
Nup145C(Q691G)-Secl3 were generated by PCR mutagenesis.
Nup145C-Sec13 was produced identically to Nup85-Sehl except that gel
filtration was performed in 10mM Hepes/NaOH pH 7.4,150mM NaCl, 1mM DTT,
0.1mM EDTA.
Full-length Nup84 from S. cerevisiae was cloned into a pET-Duet-derived
plasmid using the BamHI/NotI restriction sites. The C-terminal tail module of
Nup84 was removed from the construct to generate Nup841-42 by PCR and is
referred to as Nup84Atail in the text. Nup84-DSICD was generated by PCR
mutagenesis. The protein was purified via metal-affinity and size exclusion
chromatography as described for Nup145C-Sec13.
Full-length Nup120 from S. cerevisiae was cloned into a pET-Duet-derived
plasmid using the BamHI/Notl restriction sites. A trimeric complex between full
length Nup120, Nup85(fl), and Seh1 was produced essentially as in (Lutzmann et
al., 2002) and purified as described for Nupl45C-Secl3.
Crystallization
The Nup85*Sehl complex was crystallized in 18 % (v/v) PEG 3350, 0.2 M
sodium citrate, and 0.1 M bis-tris-propane pH 8.5 by the hanging drop vapor
diffusion method at 18*C. Crystals grew within 4-10 days forming rods with
dimensions of 150 pm x 150 pm x 400 pm. The selenomethionine derivative
crystallized in the same conditions. Native crystals were cryoprotected in
reservoir solution with 15% (v/v) PEG 400. Selenomethionine crystals were
cryoprotected in the reservoir solution supplemented with 4% (v/v) additional
PEG 3350 before flash freezing in liquid nitrogen. Both the native and
selenomethionine Nup85*Sehl complex crystallized in space group P41212 with
two molecules per asymmetric unit. Crystal screening was performed at beamline
X6A at National Synchrotron Light Source (NSLS) and final X-ray data was
collected at the NE-CAT beamline 241D-C at Argonne National Laboratory.
Structure determination
Data reduction was carried out using HKL2000 (Otwinowski and Minor, 1997).
The structure was solved with the single anomalous dispersion (SAD) technique
using the selenomethionine derivative. The positions of 2*16 selenium sites (out
of 2*20 possible) were found with the program SHELXD (Adams et al., 2002;
Sheldrick, 2008) and were used for phasing. The NCS-averaged, solventflattened 3.7 A experimental electron density map was of sufficient quality to
trace the backbone of most of the model. The selenium positions served as
markers to unambiguously assign the sequence for Nup85. Assigning the
sequence of Seh1 was assisted by superimposing the structure of the
homologous Sec1 3. The final model was refined against native data extending to
3.5 A. Model building was carried out with Coot (Emsley and Cowtan, 2004), for
refinement the PHENIX suite was used (Adams et al., 2002). Only few packing
interactions exist in the crystal, resulting in a relatively high Wilson B-factor of
118 A2 . Thus, B-sharpened maps were generated with CNS (Brunger et al.,
1998; DeLaBarre and Brunger, 2006) and were used to assist side chain
placement in the early stages of model building. NCS-restraints were applied
throughout the refinement process. The final model has good stereochemistry
and no Ramachandran outliers (84.1% of residues in preferred regions, 14.4% in
additional allowed regions) according to Molprobity (Davis et al., 2007). All
secondary structure elements of the 100 kDa heterodimer have been traced,
however several loops connecting either helices in the trunk and crown of Nup85
or strands in Seh1 are omitted due to poor electron density in those regions.
Analytical gel filtration
For Nupl45C-Secl3 and Nup84 binding experiments, equimolar amounts of
Nupl45C-Secl3 (or Nupl45C-ELIEA-Secl3) and Nup84 were mixed and
incubated at 4 0C for 5 minutes. Similarly, equimolar amounts of
Nup145CAtail-Sec13 and Nup84Atail (or Nup84Atail-DSICD) were mixed and
incubated at 4 *Cfor 5 minutes. Reactions (500 pl) were injected onto a
Superdex S200 10/300 column (GE Healthcare) and run in 10 mM Hepes/NaOH
pH 7.4,150 mM NaCI, 1mM DTT, 0.1 mM EDTA at a flow rate of 0.8 ml/min.
Nup85-Seh1 complex at various concentrations (1, 5, 10, and 20 mg/ml) was
loaded onto a Superdex S200 10/300 column and run in 10 mM Tris/HCI pH 8.0,
150 mM NaCI, 0.05 mM EDTA, and 0.5 mM TCEP at a flow rate of 0.8 ml/min.
In Figure 2.6A, equimolar amounts of Nup145C-Sec13 and Nup85(fl)-Sehl were
mixed and incubated at 4 *Cfor 5 minutes before injection on a Superdex200 HR
26/60 column. In Figure 2.6B, equimolar amounts of Nup120 and Nup85*Sehl
were mixed and incubated at 4 *Cfor 5 minutes before injection on a Superose 6
HR 10/300 column (GE Healthcare). In Figure 2.6C, Nup120ONup85(fl)-Seh1 was
mixed with a 2-fold molar excess of Nup145C-Sec13 or Nupl45CAtail*Secl3
and incubated at 4 0C for 5 minutes before injection on a Superdex S200
HR10/300 column. In Figure 2.7, Ni-NTA elutions of Nup120-Nup85(fl)-Seh1 coexpressed with Nup145C-ELIEA*Secl3 or Nup145C(Q691G)-Secl3 (where both
Nup120 and the Nupl45C-Secl3 variant are 6xHis-tagged) were run on a
Superdex 200 HR26/60 column. Gel filtration was carried out in 10 mM
Hepes/NaOH pH 7.4, 150 mM NaCl, 1mM DTT, 0.1 mM EDTA at the columns
recommended flow rates.
Analytical ultracentrifugation
Purified Nup85-Seh1 complex was gel-filtered in 10mM Tris/HCI pH 8.0, 150mM
NaCI, 0.5mM TCEP, and 0.05 mM EDTA immediately prior to the experiments.
Analytical ultracentrifugation experiments were carried out with an Optima XL-A
centrifuge using An50Ti (6 hole, equilibrium runs) or An60Ti (4 hole, velocity
runs) rotors.
Samples for sedimentation velocity (440 pl sample or 450 pl buffer) were loaded
into Epon-charcoal filled 2 channel centerpieces, fit with sapphire windows, and
spun at 42,000 rpm. Concentrations of 6.3, 3.2, and 0.63 pM Nup85-Sehl were
used. Sedimentation velocity data was analyzed globally to generate a c(s)
distribution using the hybrid local continuous distribution and global discrete
species model in SEDPHAT (Schuck, 2000). The data was fit from 2 to 10 s-13
with Sedanal calculated vbar = 0.7319 cm 3/g, r = 1.0182 cP, and p = 1.00472 g/
cm3 .
Samples for sedimentation equilibrium (110 pL sample or 120 pl buffer) were
loaded into Epon-charcoal filled 6 channel centerpieces, fit with quartz windows,
and spun at 13,500, 17,500, and 22,800 rpm, respectively. Two replicates of 6
concentrations (7.6, 6.3, 5.0, 3.8, 2.5, and 1.3 pM) were analyzed. Approach to
equilibrium was monitored with Winmatch. Absorbance data was collected at
280 nm at the smallest possible step size with 5 replicates per step.
Sedimentation equilibrium data (36 datasets total) was fit globally with Ultrascan
9.0 (http://ultrascan.uthscsa.edu) with a single ideal species model.
Isothermal titration calorimetry
Purified Nup145C-Sehl, Nupl45C-ELIEA-Sehl, Nupl45CAtail-Sehl, Nup84,
Nup84Atail, and Nup84Atail-DSICD were gel-filtered into 10mM Hepes/NaOH pH
7.4, 150mM NaCl, 1mM DTT, and 0.1mM EDTA immediately prior to the
experiment. Protein concentrations were determined spectrophotometrically at
280 nm. ITC was performed using a VP-ITC microcalorimeter (MicroCal,
Northampton, MA). Titrations were performed at 25 *Cby injecting 6 pl aliquots
of Nupl45C-Sehl, Nupl45C-ELIEA*Sehl, or Nupl45CAtail*Sehl at 4.8 pM into
the ITC cell containing 1.43 ml of Nup84, Nup84Atail, or Nup84Atail-DSICD at
0.4 pM. Binding stoichiometry, enthalpy, and entropy as well as the equilibrium
dissociation constant was determined by using the "single set of independent
sites" model of molecular association (MicroCal Origin 2.9; MicroCal).
CD spectroscopy
Nup145CAtail-Sec13, Nup145CAtail-ELIEA*Sec13, Nup84Atail, and Nup84AtailDSICD were purified as described above and gel filtered into 5 mM Hepes/NaOH
pH 7.4 and 150 mM NaCl immediately prior to the experiment. An Aviv Model
202 CD spectrometer was used for all experiments. CD signal at 208nm of 1.3
pM protein in a 1 mm pathlength cell was recorded at every degree during a 2580'C temperature ramp with two minutes of equilibration time at each step.
In vivo localization experiments
Strains were grown in YPD (1% yeast extract, 2% yeast peptone, 2% glucose) at
240C to an OD600 of 0.5-0.8. Cells were added to an equal volume of phosphate
buffered saline (PBS, pH 7.3) with 20 mg/ml Hoechst dye (Invitrogen) for 30
minutes at room temperature to stain DNA. Cells were harvested by
centrifugation, washed once in phosphate buffered saline (PBS) and viewed
using a Nikon E800 fluorescent microscope (Melville, NY) mounted with a
Hamamatsu digital camera (Bridgewater, NJ). Images were captured using
Improvision OpenLabs 2.0 software (Lexington, MA) with identical exposure
times for each sample. Final images were constructed using Adobe Photoshop
CS3 and Adobe Illustrator CS3 (Adobe Systems).
Yeast plasmid construction
The CEN/ARS plasmid SBYp1 15 containing the entire NUP145 gene with the
NUP145 promoter and 3' UTR was constructed by gap repair in yeast.
Sequences upstream and downstream of the NUP145 ORF (-800 to -100 and
+1 to +400) were amplified by PCR using Phusion DNA polymerase (New
England BioLabs) with primer combinations oES143 (5'aaaggatccGCAACACTTTCAATTGCATTTCTTCAA-3') with oES144 (5'tttgaattcCAAACGAGTTAATTCTTTCTAATTTTT-3') and oES145 (5'tatgaattcGACTGAAGCTAACGCTTTTGGAGTAAT-3')
with oES146 (5'-
aaagtcgacGAAAGAGATAGATTTCTGTAAGAAGGC-3'),
respectively. The PCR
products were cloned into the BamHI/Sall sites of pRS316 to make pES323.
Gap repair of EcoRI-digested pES323 resulted in full-length NUP145 in pRS316
(SBYp1 15). Plasmid SBYp1 16 (NUP145, LEU2, CEN) was constructed by
ligating a BamHlI-SaIl fragment from SBYp1 15 into pRS315 (Sikorski and Hieter,
1989). Plasmid SBYp1 17 (nup145C-ELIEA, LEU2, CEN) was constructed by
ligating a 1.5 kb Aatll-Avril fragment from pSB210 into the same site of
SBYp 116.
Strain construction
Yeast strains used in this study are listed in Table 2.2. Genomic tagging of
NUP133 and NUP84 in a NUP145/nup145::KANMX4 diploid (BY4743
background, Saccharomyces cerevisiae deletion consortium) was done by
homologous recombination (Janke et al., 2004) using pYM28 (eGFP:H/S3MX6)
as template. Strains were selected on SMM-histidine plates and verified by
western blotting with anti-GFP antibody and nuclear rim localization of the NupGFP chimeric proteins. The resulting diploids were transformed with SBYp1 15
(NUP145, URA3, CEN) to allow viable colonies following sporulation and tetrad
dissection. Haploid strains were then transformed with SBYp1 16 (NUP145,
LEU2, CEN) or SBYp1 17 (nup145C-ELIEA, LEU2, CEN), grown in medium
lacking leucine, and plated on 5-fluoroorotic acid plates (Figure 2.10).
Table 2.2 - Yeast strains used in this study
Genotype
Strain
YS221
YS222
YS223
YS224
YS225
MATaanup145::KANMX4 NUP133-GFP:HIS3MX6 metl7AO leu2AO his3A1 ura3AO
[SBYp1 16;(NUP145, LEU2, CEN)]
MATa nupl45::KANMX4 NUP133-GFP:HIS3MX6 metl7AO leu2AO his3A1 ura3AO
[SBYp1 17; (nup145C-ELIEA, LEU2, CEN)]
MATanup 145::KANMX4 NUP84-GFP:HIS3MX6 met1 7A0 leu2AO his3A I ura3AO
[SBYp1 16; (NUP145, LEU2, CEN)]
MATa nup145::KANMX4 NUP84-GFP:HIS3MX6 met17AO leu2AO his3AI ura3AO
[SBYp1 17; (nup145-ELIEA, LEU2, CEN)]
MATanup145::KANMX4 metl7AO leu2AO his3AI ura3AO [SBYpI 15;(NUP145,
URA3, CEN)]
Cell fractionation
Strains expressing Nupl33-GFP or Nup84-GFP were grown to log phase (OD600
= 0.5-0.7) in YPD at 30 C. Twenty-five OD600 units were harvested by filtration,
washed with cold water, and collected by centrifugation. Cells were pre-treated
with 100mM Tris/HCI, pH 9.4, 0.5% 2-mercaptoethanol for 10 minutes at 300 C
and spheroplasted in S buffer (40mM HEPES pH 7.5, 5 mM MgCl 2 , 1.2 M
sorbitol) containing 0.2mg/ml Zymolyase (1OOT) for 1 hour at 30 0C.
Spheroplasts were washed 3 times with S buffer, resuspended in 0.5mL lysis
buffer containing 20 mM HEPES, pH 7.5, 5 mM MgCl 2 , 1 mM PMSF and
protease inhibitor cocktail (Roche) and lysed on ice by Dounce homogenization.
A portion of the total lysate was removed (T), and the remaining lysate was
centrifuged at 16,000 xg for 30 minutes at 4 *C resulting in a soluble (S) and
pellet (P) fraction. Equal cell equivalents were resolved by SDS-PAGE,
transferred to nitrocellulose, and proteins were detected using antibody against
GFP (1:20,000) or the cytosolic protein Pgk1p (1:3,000).
Acknowledgements
We thank staff at beamlines 24-ID-C/-E at Argonne National Laboratory and X6A
at National Synchrotron Light Source for excellent assistance with data
collection, R. Sauer and T. Baker for critically reading the manuscript, G. Wink for
contributions, members of the Schwartz laboratory for discussions, and the
Biophysical Instrumentation Facility for the Study of Complex Macromolecular
Systems (NSF-0070319 and NIH GM68762) for providing instrumentation.
Supported by NIH grant GM77537 (T.U.S.), a Pew Scholar Award (T.U.S.), a
Koch Fellowship Award (S.G.B.), and a Vertex Scholarship (S.G.B.). Coordinates
and structure factors of the Nup85-Sehl crystal structure have been deposited in
the Protein Data Bank (PDB) with accession code 3EWE.
Chapter three
The structure of the scaffold nucleoporin Nup120 reveals a new and
unexpected domain architecture
This chapter was previously published as Leksa, N.C.*, Brohawn, S.G.* &
Schwartz, T.U. The structure of the scaffold nucleoporin Nup120 reveals a new
and unexpected domain architecture. Structure 17, 1082-1091 (2009).
*These authors contributed equally to this work.
N.C.L. designed, conducted and analyzed crystallographic and S. cerevisiae
experiments and wrote the manuscript; S.G.B. designed, conducted and
analyzed crystallographic and biochemical experiments and wrote the
manuscript; T.U.S. advised on all aspects of the project and wrote the
manuscript.
Abstract
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs),
enormous protein assemblies residing in circular openings in the nuclear
envelope. The NPC is modular, with transient and stable components. The stable
core is essentially built from two multiprotein complexes, the heptameric Y
complex and the Nic96 complex, arranged around an eightfold axis. We present
the crystal structure of Nup120 1.757 , one of the two short arms of the Y complex.
The protein adopts a compact oval shape built around a novel bipartite a-helical
domain intimately integrated with a p-propeller domain. The domain arrangement
is substantially different from the Nup85*Sehl complex, which forms the other
short arm of the Y. With the data presented here, we establish that all three
branches of the Y complex are tightly connected by helical interactions and that
the p-propellers likely form interaction site(s) to neighboring complexes.
Introduction
The main feature that distinguishes eukaryotes from prokaryotes is the
confinement of the genetic material into a membrane-enveloped nucleus.
Because gene transcription and mRNA processing occur inside the nucleus and
protein translation is restricted to the cytoplasm, transport across the doublelayered nuclear envelope (NE) is essential for cellular homeostasis. The
exchange of all molecules, including ions, proteins, and RNAs, is facilitated
exclusively by nuclear pore complexes (NPCs) (D'Angelo and Hetzer, 2008; Lim
et al., 2008; Tran and Wente, 2006; Weis, 2003). NPCs are large protein
assemblies of 40-60 MDa that are embedded in the nuclear envelope and exhibit
an 8-fold rotational symmetry around a central axis in addition to an imperfect
two-fold symmetry across the plane of the NE (Beck et al., 2007; Stoffler et al.,
2003)(Beck et al., 2007; Stoffler et al., 2003). Composed of multiple copies of ~
30 proteins, termed nucleoporins (nups), the NPC has an outer diameter of -100
nm whereas the central channel measures -40 nm in width. Transmembrane
nups directly connect the NPC to the NE, while the phenylalanine-glycine (FG)
repeat-containing Nups line the interior of the pore. These FG-filaments mediate
nucleocytoplasmic transport of cargo molecules across the NE. FG-filament
bearing nups are anchored to the NPC scaffold built from architectural
nucleoporins arranged in two large multiprotein complexes that form a
membrane-proximal layer. The scaffold structure is very stable and undergoes
virtually no turnover in the quiescent cell (D'Angelo et al., 2009), whereas many
other nucleoporins have variable dwell times at the NPC (Rabut et al., 2004). In
consequence, the NPC is a highly modular structure (Schwartz, 2005).
Understanding the structure of the NPC therefore depends upon elucidating its
basic scaffold.
The two essential architectural building blocks of the NPC are the Nup84 or Y
complex and the Nic96 subcomplex. The components of the Nic96 subcomplex
likely include Nic96, Nup53/59, Nup157/170, Nup188 and Nup192 (yeast
nomenclature), as inferred from co-immunoprecipitations (co-IPs) (Alber et al.,
2007b; Hawryluk-Gara et al., 2008; Marelli et al., 1998; Onischenko et al., 2009)
and yeast-two-hybrid screens (Wang et al., 2009; Yu et al., 2008). Judged by
immunolabeling, the Nic96 subcomplex might form a central ring within the NPC
sandwiched between peripheral rings formed by Y complexes (Alber et al.,
2007b). In comparison to the Nic96 subcomplex, the Y complex is substantially
better understood. It has 7 universally conserved members
(yeastNup84/humanNupl07, yNup85/hNup75, yNupl20/hNupl60, Nup133,
yNupl45C/hNup96, Sec13, and Seh1) and three additional members (Nup37,
Nup43, and ELYS/Mel-28) to date found mainly in metazoa (Cronshaw et al.,
2002; Gillespie et al., 2007; Loiodice et al., 2004; Rasala et al., 2006). In the
fungus Aspergillus nidulans, distant Nup37 and ELYS orthologs have been
described recently (Liu et al., 2009). The heptameric core Y complex assembles
tightly as shown by co-IPs and in vitro assembly (Harel et al., 2003; Lutzmann et
al., 2002; Siniossoglou et al., 2000; Walther et al., 2003). Negatively-stained
electronmicrographs of the assembled Y complex reveal a branched Y-shaped
structure, with two short arms and a kinked stalk connected at a central hub
(Lutzmann et al., 2002).
Crystallographic analysis of the Y complex has progressed quickly. The kinked
stalk ends with a flexibly attached p-propeller domain (Berke et al., 2004) at the
N-terminus of Nupl33 followed by an irregular C-terminal helical stack domain
that connects end-to-end to Nup84 (Boehmer et al., 2008; Whittle and Schwartz,
2009). The Nup84*Nupl33 interface defines at least one kink in the stalk. The
opposite end of Nup84 links to Nup145C (Brohawn et al., 2008).
Nupl45C*Secl3 (Hsia et al., 2007) resides proximal to the hub (Lutzmann et al.,
2002). Seh1 -Nup85 forms one of the two short arms of the Y complex (Brohawn
et al., 2008; Debler et al., 2008). Nup84, Nup85, and Nup145C are structurally
related (Brohawn et al., 2008), despite very low sequence conservation, as are
the p-propeller proteins Seh1 and Sec13.
Nup120 is the last remaining Y complex component without structural
information. Here we report the 3.0 A crystal structure of Nup120 (residues 1-757
of 1037), which reveals a compact and rigid structure composed of an N-terminal
p-propeller domain tightly integrated into a novel bipartite a-helical domain. Our
structure largely defines the second short arm of the Y complex. Comparison
with other members of the Y complex, phylogenetic analysis, in vitro binding
experiments, and in vivo localization data suggest a role for Nup120 consistent
with our lattice-like model of the NPC.
Results
Structure determination
After systematic C terminal truncation, a stable fragment comprising most of
Nup120 (residues 1-757 of 1037 total) from S. cerevisiae was recombinantly
expressed in E. coli and purified. The protein is a monomer in solution (data not
shown). Native protein readily crystallized and selenomethionine derivatized
crystals were obtained after microseeding with native crystals. Though both
crystal forms were optically identical, the selenomethionine crystals diffracted
better and were used exclusively in structural analysis. The structure of Nup120
was solved with one molecule per asymmetric unit by single-wavelength
anomalous dispersion (SAD) on very strong Se-Peak data (all 9 Se sites are well
ordered). The model is complete except for 26 residues at the C-terminus and
three flexible loops and was refined to Rwork / Rfree of 24.4 % / 29.9 % (Table
3.1).
Table 3.1 - Data collection and refinement statistics
Nup1201.757
Selenomethionine
Data collection
Space group
Cell dimensions
a, b, c (A)
a, #, y (0 )
Resolution (A)
Rsym (%)
P21212
114.6, 153.7, 53.0
90,90,90
50-3.0 (3.1-3.0) *
5.1
//al
20.4 (1.8)
Completeness (%)
Redundancy
99.8 (99.6)
3.0 (3.0)
Refinement
Resolution (A)
50.0
No. reflections
Rwork
/ Rfree
No. atoms
Protein
Water
B-factors (A2)
B-Propeller
a-helical insertion
a-helical domain
Ramachandran plot (%)
Favored/allowed/outliers
RMSD bond lengths (A)
-
3.0
35841
24.4 / 29.9
5305
0
114
89
83
93.6 / 5.3 / 1.1
0.017
RMSD bond angles (0)
1.915
*Values in parentheses are for highest-resolution shell.
Crystal structure of Nup120
Nup120 folds into a continuous, prolate disk with overall dimensions of 90 A x 55
A x 35 A. One half of the structure is formed by an N-terminal p-propeller domain
that is intimately connected to a compact central domain built from two closely
packed a-helical segments (Figure 3.1). Overall, the structure is better resolved
in the a-helical segment than the p-propeller, likely a result of a paucity of
packing contacts involving the latter. The p-propeller of Nup120 contains 7
consecutive blades that fan out from a central axis. The blades are formed by a
p-sheet of 4 consecutive antiparallel strands, labeled A-D. Blade 7 is built from
the very N-terminus of the polypeptide chain forming strand 7D and joining
strands 7A-C to close the propeller in a Velcro-like closure commonly observed
in p-propeller domains (Chaudhuri et al., 2008). Blade 1 is 5-stranded, with
strand 7D extending to form the additional strand 1E before connecting to strand
1A (Figure 3.1 E). Blade 3 is somewhat unusual in that the outermost strand 3D is
only loosely connected to strand 3C with a hydrogen-bonding network hardly
visible in our structure.
Nupl20
wbertion
]
9o
35A
Figure 3.1 - Overall topology of Nup120
(A) Current model of the Y complex. The relative position of Nup1 20 is highlighted.
(B) Schematic of full-length Nup1 20 from S. cerevisiae. Residues that form the p-
propeller are colored blue, those that form the a-helical domain are purple, and
those not present in the crystallized construct are in gray. (C,D) The overall
topology of Nup1 20 (residues 1-757 of 1037) is shown in two views rotated by 90'.
The structure is gradient-colored from blue to white to magenta from N-to Cterminus. At its N-terminus, Nup120 forms a 7-bladed p-propeller. A 4-helix bundle
(al -a4) between blades 6 and 7 packs against the remainder of the helical domain
(a5-al5), composed of helices wrapping around a central hydrophobic stalk of the
two long helices al1 and al2. Unstructured loops absent from the final model are
shown in gray. (E)A topological diagram of the Nup120 structure is shown,
illustrating the 4-helix insertion between blades 6 and 7 of the propeller as well as
the two central helices of the helical domain.
The a-helical domain that forms the second half of the molecule is constructed in
a unique discontinuous manner. In total the domain contains 15 helices, labeled
al-al5. The first 4 helices form a compact bundle and are inserted between
blades 6 and 7 of the p-propeller. The remaining 11 helices are C-terminal to the
p-propeller and pack tightly against the 4-helix bundle to form one compact
entity. The arrangement of the helices is highly irregular. The most prominent
feature of the domain are two long helices, al1 and a12, which pack against
each other and form a central stalk, defining the long axis of the domain. Helices
a5-a9 wrap up and around this element, with helices a6/a7 and a8/a9 arranged
in two stacked braces oriented perpendicular to the stalk. Helices al, and a13a15 meander back down and around the other side to bury most of the
hydrophobic stalk. The remaining surface area of the two central helices is
closed by the 4-helix insertion bundle. Taken as a whole, the structure of
Nup120 .1 757 consists of a bipartite helical domain that is interrupted by a ppropeller.
The main crystal contact is formed by a domain swap
Other than a collection of spurious small contacts, crystal packing is mainly
achieved by a domain swap of the terminal helices a15 and a15' exchanging
between two neighboring molecules (Figure 3.2A). The interface measures
1355 A2 , is entirely hydrophobic and highly complementary (Figure 3.2B).
Domain swaps are regularly found in crystals (Liu and Eisenberg, 2002) and, as
stated above, we do not observe dimerization of Nup120 in solution. We cannot
rule out the possibility that the interface is physiologically relevant; sterically the
domain swap is conceivable in the context of the entire molecule including the Cterminal 280 residues omitted in our construct. It is however more likely that the
exposed hydrophobic patch is artificially generated by the truncation of the
domain, since we also do not observe particularly high sequence conservation
within helix al5. We speculate that in vivo the patch likely accommodates one of
the additional helices from the C-terminal domain, or alternatively, is involved in
interaction with a neighboring molecule. Whether the C-terminal domain is rigidly
or flexibly tethered to Nup 1201.757 is an open question.
A
B
Nup12
C-ter
Figure 3.2 - Crystal contacts between two symmetry-related molecules
(A) One molecule of Nup120 in blue, and its symmetry mate, related by a 2-fold
rotation, in orange. The p-propellers are at opposite ends while the helical
domains engage in a putative domain swap between helices al 5 of both
molecules. (B)Close-up of the domain-swapped region, illustrating the
hydrophobic nature of helix al 5 and the surrounding pocket (hydrophobic residues
are shown in white). In monomeric Nup120, helix a15 likely folds under (arrow)
and occupies the position taken by helix al 5' (orange) of the symmetry-related
molecule in the crystal.
Conservation of Nup120 and comparison to the human ortholog Nup160
Overall, sequence conservation between Nup120 orthologs is weak as is
typically observed in scaffold nucleoporins (Brohawn et al., 2008; Jeudy and
Schwartz, 2007). Most of the better-conserved residues are buried in the
hydrophobic core of the protein and are involved in maintaining the structural
integrity of the protein. On the protein surface we find few conserved patches
(Figure 3.3A). Most distinct is an area on the edge of the p-propeller,
corresponding to the outer strands of blade 3 and the loop leading into blade 4.
The conserved sequence begins in the 3BC loop and continues into strand 3C
itself. Although generally buried in canonical p-propellers, here strand 3C is quite
exposed. This is probably the result of weaker interactions with strand 3D, which
is flanked by two large loops and peels away from the core of the propeller.
Additional conserved residues are spotted around this area, creating a relatively
large conserved patch. The potential significance of this observation is discussed
below.
A
180*
Bottom View
Top View
P-Propeller View
Top View
P-Propeller View
I8o~
Bottom View
Figure 3.3 - Surface conservation and electrostatics of Nup120
(A) Surface conservation of Nup120 is shown from three different views. To
illustrate the conservation of residues on the surface of Nup120, a multiple
sequence alignment sampling the phylogenetic tree of budding yeasts was
generated and mapped onto the surface, colored from white (not conserved) to
orange (highly conserved). The view in the middle panel corresponds to the view
shown in Figure 3.1C. A patch of highly conserved residues is apparent on the
outer face of the propeller domain of Nup1 20. (B) The electrostatic surface
potential of Nup120 is shown in the same views as in (A) and is colored from red (10 kT/e) to blue (+10 kT/e).
We analyzed the charge distribution on the surface of Nup120 (Figure 3.3B).
Since Nup120 is part of the scaffold structure of the NPC, we asked whether it
may be possible that it directly juxtaposes the pore membrane. This would also
be consistent with a membrane-curvature sensing ALPS motif, predicted in helix
a5- a6 of Nup120 (Drin et al., 2007). The surface charge of Nup1201.77,
however, is fairly mixed without conserved positive patches that might suggest
direct membrane interaction. The ALPS motif is deeply embedded in the
structure and it is rather unlikely that it would swing out and insert in the
membrane. Thus we suggest that Nup120 1. 757 does not directly touch the nuclear
membrane.
Structure-guided sequence comparison of Nup120 and its human ortholog
Nup160 strongly suggests that both proteins adopt the same unique fold despite
a low sequence identity of -10 %. Both non-canonical characteristics of Nupl20
(the helical insertion between blades 6 and 7 of the N-terminal p-propeller and
the long central stalk helices forming the hydrophobic core of the central domain)
are clearly conserved in Nup160. The 279 additional residues of Nup160 are
dispersed over several regions and mostly correspond to different loop lengths
connecting a-helices and p-strands. Of note, the C-terminal domain of Nup160,
which is not present inthe Nup120 crystal structure described here, has 5
additional predicted helices, possibly indicating a vertebrate-specific extension.
Despite these differences, the Nup120 crystal structure is likely generally
representative of all Nup120/Nup160 orthologs.
The C terminus of Nup120 directly binds Nup145C and Nup85
We sought to map the interaction of Nup120 with its direct binding partners in the
Y complex, Nup145C and Nup85. In a gel filtration assay, we tested for the
formation of a pentameric Sec13-Nupl45C*Nupl20-Nup85*Seh1 complex
(Figure 3.4). Incubating Nup120766 -1037 or Nup120 1.757 with both Nup145C* Sec13
and Nup85-Seh1 resulted in complex formation only for the C-terminal Nup120
domain, but not for the crystal construct. In combination with previous interaction
mapping experiments (Brohawn et al., 2008), we conclude that the helical tails of
the ACE1 domains of both Nup145C and Nup85 each interact directly with the
helical Nup120 766 -10 37 . This positions the C-terminus of Nup120 at the center of
the hub of the Y complex.
A
10
13
16
Elution Volume (mL)
Elution volume (mL)
i
"
aM so
-
.0
1-."U14C
pn
hi
p120 (766-1037)
Figure 3.4 - The C-terminus of Nup120 is necessary for binding
Nup85eSehl and Nupl45C*Secl3
(A) Nup85*Sehl (red), Nupl45CeSecl3 (gray), and Nup1201.7y57 (green) were
run individually and in combination (blue) on a Superdex S200 10/300 gel
filtration column. (B) Nup85*Sehl, Nupl45C*Secl3, and Nup120766 -1037 were
incubated together and run on Superdex S200 26/60 and eluted in a single peak.
(C) Fractions from the gel filtration experiment in B were analyzed by SDS-Page.
Co-migration of Nup85-Sehl, Nup145C-Sec13, and Nup120 76610 3 7 indicates that
the C-terminus of Nup1 20 is necessary for the formation of the pentameric
complex that comprises the hub of the Y complex.
Without its C-terminal domain Nup120 does not properly localize to the
NPC
Having established that Nup120 766 . 103 7 is sufficient to bind both Nup145C-Secl3
and Nup85*Sehl in vitro, we sought to examine the integration determinants of
Nup120 into the NPC in vivo. NUP120 is not essential in yeast but nup120A cells
exhibit a pore clustering phenotype (Aitchison et al., 1995; Heath et al., 1995)
that is reminiscent of, but less severe than, the pore clustering observed for other
scaffold nucleoporins including Nup84 and Nup133 (Li et al., 1995; Pemberton et
al., 1995; Siniossoglou et al., 1996). We genomically GFP-tagged full length
Nup120 and replaced the C-terminal 280 residues of genomic Nup120 with an in
frame GFP-tag to create strains expressing Nupl20-GFP or Nupl201..75-GFP in
a BY4741 background and examined the localization of the proteins via
immunofluorescence (Figure 3.5). Nupl20-GFP properly localizes to the NPC
and shows typical nuclear rim staining, superimposing well with mAb414-staining
of FG-Nups (Aris and Blobel, 1989). Nup120 1. 757-GFP, on the other hand, does
not properly localize to the nuclear envelope and shows staining throughout the
cell. This result is consistent with our in vitro data and suggests that the
integration into the Y complex is important for proper localization of Nup120.
nup84A
mT
nup133A
nup 120A
Nup1 20 1-7srGFP
Nupl20 1-1037GFP
(P
-Q
*2k
0~
3
-o
-n
0
Figure 3.5 - Nup120 1757 does not localize to the nuclear envelope
(Aa-Ad) Nupl20-GFP is targeted to the nuclear envelope, as confirmed by colocalization with mAb414 (staining FG-Nups), while Nup1201 7. 57-GFP (Ba-Bd) is
distributed throughout the cell. Mislocalization indicates that the C-terminus of
Nup120 is necessary for proper recruitment to the NPC. Nup120A, nup133A,
and nup84A cells (in the same BY4741 strain background) are shown for
comparison (Cb-Cd,Db-Dd,Eb-Ed). Nuclear rim was visualized using mAb414,
GFP-tagged Nup120 using goat a-GFP, and DNA using DAPI. Merged images
are shown on the right.
Nup120 is topologically different from other scaffold nucleoporins
A recent surge in the X-ray crystallographic analysis of components of the NPC
has greatly increased the repertoire of available structures of nucleoporins
constituting the structural scaffold of the NPC. These structures (including those
of Nic96 (Jeudy and Schwartz, 2007; Schrader et al., 2008b), Nupl33-NTD
(Berke et al., 2004), Nupl33*Nupl07 interaction complex (Boehmer et al., 2008),
Nupl45C*Secl3 (Hsia et al., 2007), and Nup85-Sehl (Brohawn et al., 2008;
Debler et al., 2008) as well as associated biochemical experiments, have led to a
deeper and broader understanding of how the scaffold of the NPC is assembled
from its constituent parts.
The structural subunits of the NPC were initially predicted to be composed of
simple combinations of regular a-helical solenoids and p-propellers (Devos et al.,
2006). Experimental data now allows us to specify these broad classifications,
which should help to more specifically address the ancestry of the NPC. Both
Secl3 and Seh1 form open, 6-bladed propellers that are completed in trans by
the N-terminal insertion blades of their binding partners Nup145C and Nup85,
respectively. Furthermore, helical nucleoporins Nic96, Nup145C, Nup85, and
Nup84 are built around a common and distinct ancestral coatomer element
(ACE1) shared with Sec3l of the outer coat of COPII vesicles (Brohawn et al.,
2008). InACE1 proteins, a specific N-terminal elaboration is followed by a
tripartite helical domain composed of a trunk, a crown, and a tail module. The ~
30 helices within ACE1 follow a J-like pattern, zigzagging up on one side of the
trunk, making a U-turn within the crown domain, and then following down on the
opposite side of the trunk (Figure 3.6A, right panel). The tail module is often
attached with modest flexibility to the trunk and is missing in most crystal
constructs. In the case of Nup145C and Nup85 the N-terminal elaborations are
the aforementioned insertion blades that bind to Secl3 and Seh1.
Nupl45C*Secl3 and Nup85*Sehl heterodimers form the two proximal
segments of the Y complex and are tethered together by Nupl20 (Brohawn et
al., 2008).
Based on structure predictions and its overall size, it was reasonable to suggest
that Nupl20 may take on a structure similar to Nupl45C*Secl3 and
Nup85eSehl, with the only major difference being that the p-propeller and the ahelical domains are fused into one polypeptide chain. However, comparison
between the structure of Nupl20 and the Nup85*Sehl heterodimer reveals a
marked difference in topology (Figure 3.6A). Whereas the ACE1 architecture of
Nup85 forms an elongated a-helical domain, the central a-helical domain of
Nupl20 is nearly as wide as it is long, forming an almost globular structure. The
ACE1 trunk module covers the bottom face of the Seh1 p-propeller, while in
Nupl20 the helical domain is attached to and integrated into an edge of the ppropeller. Further, the ACE1e p-propeller interaction is accomplished by the
addition of an insertion blade N-terminal to ACE1, while in Nupl20 the ppropeller domain inserts a 4-helix bundle into the central a-helical domain. This
helical insertion fits snugly into a pocket formed by helices a5-a7 and al 1-a13
and buries a surface of nearly 600 A2 (Figure 3.6B).
The extensive interaction between the p-propeller and a-helical domain of
Nupl20 buries a total surface area of 2175 A2 and creates a rigid interface. In
contrast, the largest buried surface area between ACE1 and its p-propeller
partner is at the insertion blade/p-propeller interface. Additional contact areas in
ACE1 *p-propeller complexes are smaller in comparison to the corresponding
interfaces in Nupl20 and, importantly, far less hydrophobic. Thus, for the
ACE1-p-propeller assembly one has to consider substantial flexibility about the
interaction joint, while the Nup120 structure presented here is very likely
inflexible. Not only does the structure of Nup120 significantly differ from the
ACE1- p-propeller heterodimers, but additional emerging evidence suggests that
it also lacks similarity to Nup170 and Nup133, the two other scaffolding
nucleoporins of similar size and domain composition with an N-terminal
propeller followed by an a-helical domain (Whittle and Schwartz, 2009).
p-
insertion
bundle
Nup120
ACE1
B
insertion
bundle5
825 A215o
A2
Figure 3.6 - Nup120 is composed of a combined p-propeller - a-helical
domain distinct from ACE * P-propeller
(A)The overall architectures of Nup120 and the ACE1 motif of Nup85-Sehl are
distinctly different. Nup1 20 is characterized by a bipartite helical domain (blue to
white from N-to C-terminus) that is interrupted by a p-propeller domain (gray).
The Nup85 ACE1 motif is characterized by an elongated helical stack (colored blue
to white from N-to C-terminus) that makes a U-turn in the crown domain of the
molecule. At its N-terminus, Nup85 inserts a blade (in red) into the open, 6-bladed
Seh1 p-propeller. Incontrast, the p-propeller of Nup120 contributes a helical
insertion bundle (red) to the helical domain. The view of Nupl20 is the same as
that in Figure 3.3B. (B) Surface representations of intact Nup120 are shown on the
100
left, while on the right the three modules of Nup1 20 - the propeller, the helical
insertion, and the helical domain - are shown pulled apart to illustrate the buried
surface areas in between. Interacting surfaces between the propeller and the
insertion bundle are outlined in green, between the propeller and the helical
domain in yellow, and between the insertion bundle and the helical domain in
orange. The molecule is N-to-C gradient-colored from blue-to-white-to-magenta.
Discussion
Here we report the crystal structure of Nup120, a large, universally conserved
architectural nucleoporin. This structure adds substantially to the growing
inventory of crystallographically characterized nucleoporins. As a result of these
studies, we learn that the NPC is constructed from nucleoporins with a limited set
of domain architectures. While other a-helical and p-propeller domains of scaffold
nucleoporins fall into distinct classes, likely pointing to gene duplication in the
early evolution of the NPC, the Nup120 architecture appears to be quite distinct.
A search for structurally related proteins fails in detecting similarity beyond the
isolated p-propeller scaffold or the arrangement of more than 6 a-helices. Within
the list of crystallographically uncharacterized nucleoporins, none is likely to
match the Nup120 structure closely.
Nup120 in the context of the NPC scaffold
Nup120 forms one of the two short arms of the universally conserved, 0.6 MDa Y
complex, the essential building block of the NPC scaffold. The assembly of the Y
complex from its 7 members is fairly well understood and has been studied using
many different techniques. All of these studies profit from generally very high
affinities observed between the interacting proteins within the Y, which generated
largely consistent co-immunoprecipitation and yeast two-hybrid results and
facilitated the crystallization of several complex crystal structures. However,
models for the assembly of the NPC structural scaffold and the integration of the
Y complex are still controversial.
101
Based on a combination of computational, structural, biochemical, and in vivo
experiments, a model was proposed where the Y complex is positioned in two 8membered rings located at the periphery of the NPC sandwiching two equally
wide rings composed of Nupl57/170,Nupl88 and Nupl92 in between (Alber et
al., 2007b).
Blobel and coworkers proposed a concentric cylinder model based on crystalpacking interactions where four 8-membered rings of the Y complex are stacked
and placed directly adjacent and in contact to the curved membrane (Debler et
al., 2008; Hsia et al., 2007). Further, SehlNup85 and Sec13*Nup145C are both
supposed to form heterooctameric fence poles spanning the NPC vertically,
thereby connecting the 4 stacked rings. Nup157/170, Nup188, Nup192 and
Nic96 are suggested to form a second inner layer bridging to a third layer
composed of FG-nups. With a scaffold twice the mass of the computer-generated
model, the concentric cylinder model generates a densely packed NPC coat.
In contrast, we proposed a lattice-like model for the NPC, extrapolated from the
assembly of COPII vesicle coats and substantiated by the structure and
assembly principles of core components of the NPC scaffold (Brohawn et al.,
2008; Brohawn and Schwartz, 2009a). We propose the Y complex does not
directly coat the pore membrane (in analogy to the COPIl outer coat), but is
anchored by another set of proteins, likely involving the essential transmembrane
nucleoporin Ndcl and/or its direct binding partners (Onischenko et al., 2009). It is
of interest to discuss this issue in respect to the membrane-inserting ALPS motif
that was experimentally characterized within a loop structure in hNupl33-NTD
and that was predicted to occur as well in yNup85 and yNupl20 (Drin et al.,
2007). Based on the structural data now available on both yNup85 and yNup120,
it appears unlikely that the predicted ALPS motif in both proteins is functional in
membrane-binding since neither is in an exposed region of the protein, or is likely
to become exposed. This is in contrast to the ALPS motif in hNupl33-NTD,
where it is well exposed in the crystal structure, and also highly conserved in
102
metazoa (Berke et al., 2004). Taking all the available data together, it appears
more reasonable to suggest a specific function for the ALPS motif in metazoan
Nup1 33 rather than a general function in anchoring of the NPC to the pore
membrane. Since Nup133-ALPS is only poorly conserved in yeast, it is tempting
to speculate that it may have a specific role in NPC assembly in open mitosis
(Guttinger et al., 2009).
We predict that the lattice scaffold of the NPC is built from edge and vertex
elements, following similar assembly principles as established for COPII.
However, in the absence of definitive inter-subcomplex interaction data, any
detailed NPC assembly model is still premature and has to be interpreted
cautiously.
The fact that inter-subunit interactions are still obscure suggests that these
interactions are rather weak and hard to establish. Each short arm of the Y
complex contains one p-propeller domain, while the stalk contains two (Figure
3.1). For the assembly of the extensions of the Y, direct interactions between the
a-helical domains is essential, however this does not exclude the participation of
the p-propellers. It is reasonable to suggest that the p-propellers are prime
candidates for the elusive inter-subcomplex contacts. The vertices of the outer
coat of COPII vesicles are assembled exclusively via p-propeller interactions,
which have still only been inferred by fitting crystal structures into EM maps (Fath
et al., 2007; Stagg et al., 2008). p-propellers make excellent protein-protein
interfaces due to their inherent ability to pair with a binding partner in multiple
modes. Binding to peptides via the face of the p-propeller is well known (Jawad
and Paoli, 2002). Additionally, each blade exposes on its edge (typically on
strand D)a stretch of ~ 6-8 residues available for intermolecular p-sheet
formation, which can be likened to one half of a zipper. In Nup120, 5 of the 7
blades are exposed this way, two are buried in the hydrophobic core shared with
the attached a-helical domain. In addition to these interactions being relatively
weak, another inherent difficulty in identifying them is that they are likely very
103
poorly conserved at the sequence level because the contacts are mediated via
the backbone rather than side chains. Based on the available data, it is
conceivable that the Nup120 p-propeller is involved in inter-Y complex contacts.
It is also possible that it is used to bridge to the Nic96 complex, but we can also
not exclude that it may be an anchor for dynamic nucleoporins or other
accessory proteins. The relatively mild nup120A phenotype (Figure 3.5)
compared to nupl33A or nup84A and the behavior of Nupl20 1.-757-GFP suggests
that if the Nup120 p-propeller has an integral role in the NPC scaffold, it is either
redundant or can be functionally replaced by another nucleoporin.
In summary, we show that Nup120 adopts a unique architecture to build one of
the two arms of the multimeric Y complex, the linchpin of the NPC scaffold. The
atomic structure of the universally conserved heptameric core of the Y complex is
now nearing completion. With reliable data on inter-subcomplex contacts the
construction of a basic NPC architecture is within reach in the close future.
Methods
Protein expression and purification
Nup120 from S. cerevisiae (residues 1-757 of 1037) was expressed at 180C in
E. coli strain BL21(DE3)-RIL as a 6xHis N-terminal fusion protein from a pET-
Duet-derived plasmid. Cells were pelleted and resuspended in lysis buffer (50mM
potassium phosphate pH 8.0, 500mM NaCI, 40mM imidazole, 5mM pmercaptoethanol). Cells were lysed using a French press and the cleared lysate
incubated in batch with Ni-affinity resin. After washing the resin in batch with lysis
buffer, the protein was eluted with lysis buffer containing 250mM imidazole. After
cleavage of the purification tag, Nup120 was subjected to size exclusion
chromatography on Superdex S200 equilibrated in 10mM Tris/HCI pH 8.0,
150mM NaCI, 0.1mM EDTA, and 1mM DTT. Nup120 eluted as a monomer of 88
kDa. Selenomethionine-derivatized protein was prepared as previously
104
described (Brohawn et al., 2008) and Nup120-SeMet was purified identically to
the native version.
Full length Nup85 in complex with Seh1 and a single chain version of full-length
Nup145C in complex with Sec1 3 from S. cerevisiae were cloned as described
(Brohawn et al., 2008), purified as for Nup120 (residues 1-757), and are referred
to in the text as Nup85*Sehl and Nupl45C-Secl3. The C-terminal helical
domain of Nup120 (residues 766-1037) was generated from a full length Nup120
construct by PCR. A 5-protein complex of Nup120 (residues 766-1037),
Nup85*Sehl, and Nup145C-Sec13 was prepared by co-expression of a trimeric
complex of Nup120 (residues 766-1037)-Nup85-Seh1 (Brohawn et al., 2008) and
the single chain version of Nup145C*Secl3 in BL21(DE3)-(RIL) cells and was
purified as for Nup120 (residues 1-757). The Ni-NTA elution was pooled,
digested with human rhinovirus 3C to remove fusion tags, and subjected to size
exclusion chromatography using a Superdex S200 26/60 column equilibrated in
10mM Tris/HCI pH 8.0, 250mM NaCI, 1mM DTT, and 0.1mM EDTA.
Protein crystallization
Nup120 concentrated to 20 mg/ml was crystallized in 15% (w/v) PEG 3350, and
0.1M Tris/HCI pH 7.5, 0.2M KSCN by the hanging drop vapor diffusion method at
180C in 2pl drops. Crystals grew within 3-6 days forming rhomboid prisms with
dimensions of 60pm x 60pm x 20pm. The selenomethionine derivative
crystallized in the same condition, while the highest quality crystals were
obtained by microseeding with native crystals. Both native and derivative
crystals were cryo-protected by serial transfer of the crystals into reservoir
solutions supplemented with increasing amounts of PEG200 (10%-25% (v/v), 5%
steps) before flash freezing in liquid nitrogen. Both native and derivative protein
crystallized in space group P21212 with one molecule per asymmetric unit. Data
was collected at beamline 241D-C at Argonne National Laboratory.
105
Structure determination
Although the native crystals were larger and optically superior, the
selenomethionine-derivatized crystals diffracted significantly better and were
exclusively used for data analysis. A complete dataset was collected at the SePeak wavelength and data reduction was carried out using the HKL2000
package (Otwinowski and Minor, 1997). All 9 selenium sites were found using
SHELXD (Sheldrick, 2008). After refinement of the Se positions and density
modification with SHARP, an excellent experimental electron density map was
obtained, allowing for the assignment and building of the majority of the structure.
Sequence assignment was aided by using the selenium positions as markers.
Model building was done with Coot (Emsley and Cowtan, 2004) and refinement
was carried out using the PHENIX suite (Adams et al., 2002). The model is
complete except for residues 31-52, 188-200, 303-313, and 731-757 for which
only spurious electron density was observed. Blades 3 and 4 of the p-propeller
have the highest temperature factors and are not as well packed as the
remainder of the molecule.
Analytical size exclusion chromatography
For Nup120 (residues 1-757), Nup145C-Sec13, and Nup85-Sehl binding
experiments, equimolar amounts of each component were incubated alone or in
combination for 30 minutes at 40C in binding buffer (10mM Tris/HCI pH 8.0,
250mM NaCI, 1mM DTT, 0.1mM EDTA). Reactions were injected onto a
Superdex S200 HR 10/300 column (GE Healthcare) equilibrated in binding
buffer, and run at a flow rate of 0.8 ml/min (Figure 3.4A).
Yeast strain construction
Deletion strains were taken from the Yeast Deletion Consortium (Winzeler et al.,
1999), C-terminal GFP-tagging was done by homologous recombination in a
BY4741 background, using pFA6a-GFP(S65T)-kanMX6 as template for Cterminal modifications (Longtine et al., 1998). Strains were selected on G418
plates (200 pg/ml) and verified by PCR.
106
Fluorescence microscopy
Strains were grown overnight in YPD (1% yeast extract, 2% yeast peptone, 2%
glucose) at 300C, diluted 20-fold into fresh YPD, and grown for 4-5 hours at 300C
to OD600 -0.5. Cells were harvested by centrifugation, fixed for 3 minutes in
3.7% formaldehyde/0.1M potassium phosphate pH 6.5, and prepared for
immunofluorescence as previously described (Kilmartin and Adams, 1984).
Samples were incubated with mAb414 (abcam, 1:1000) alone or in combination
with goat anti-GFP (1:500) for 90 minutes at room temperature. Bound
antibodies were detected by incubation with Cy5-conjugated anti-mouse (1:500)
alone or in combination with Cy2-conjugated donkey anti-goat (1:200) for 45
minutes at room temperature. DNA was stained with 0.05 pg/mI 4',6-diamidino2-phenylindole (Sigma-Aldrich) and samples were mounted for imaging in 1
mg/ml p-phenylenediamine and 90% glycerol. Fluorescence microscopy was
performed on a Zeiss Axiolmager.Z1 microscope and images were taken with a
Zeiss AxioCam HRm camera.
Accession number
The atomic coordinates of the Nup120 structure described here will be deposited
with the Protein Data Bank (PDB) under the accession code 3HXR.
Acknowledgements
We thank staff at the NE-CAT beamline 24 at Argonne National Laboratory for
excellent assistance with data collection, lain Cheeseman for providing
antibodies, Eric Spear for advice on in vivo experiments, and Dennis Kim for use
of his fluorescence microscope. Supported by NIH grant GM77537 (T.U.S.), a
Pew Scholar Award (T.U.S.), a Koch Fellowship Award (S.G.B.), and a Vertex
Scholarship (S.G.B.).
107
Chapter four
Molecular architecture of the Nup84-Nupl45C*Secl3 edge element
in the nuclear pore complex lattice
This chapter was previously published as Brohawn, S.G. & Schwartz, T.U.
Molecular architecture of the Nup84-Nupl45C*Secl3 edge element in the
nuclear pore complex lattice. Nature Structural & Molecular Biology 16, 11731177 (2009).
S.G.B. designed, conducted, and analyzed biochemical, biophysical, and
crystallographic experiments and wrote the manuscript; T.U.S. advised on all
aspects of the project and wrote the manuscript.
108
Abstract
Nuclear pore complexes (NPCs) facilitate all nucleocytoplasmic transport. These
massive protein assemblies are modular, with a stable structural scaffold
supporting more dynamically attached components. The scaffold is made from
multiple copies of the heptameric Y complex and the heteromeric Nic96 complex.
We previously showed that members of these core subcomplexes specifically
share an ACE1 fold with Sec3l of the COPII vesicle coat, and we proposed a
lattice model for the NPC based on this commonality. Here we present the crystal
structure of the heterotrimeric 134-kDa complex of Nup84-Nup145CeSec13 of
the Y complex. The heterotypic ACE1 interaction of Nup84 and Nup145C is
analogous to the homotypic ACE1 interaction of Sec3l that forms COPII lattice
edge elements and is inconsistent with the alternative 'fence-like' NPC model.
We construct a molecular model of the Y complex and compare the architectural
principles of COPII and NPC lattices.
Introduction
Ineukaryotic cells, physical separation of the nucleus and cytoplasm by the
nuclear envelope necessitates a conduit for nucleocytoplasmic molecular traffic.
This gateway is solely provided by NPCs, proteinaceous channels that stud the
nuclear envelope (Brohawn et al., 2009; D'Angelo and Hetzer, 2008; Tran and
Wente, 2006; Weis, 2003). NPCs are among the largest assemblies in the cell, at
-50 MDa, and have crucial roles in cellular homeostasis. The overall structure of
the NPC is generally conserved across species (Elad et al., 2009). NPCs have a
central scaffold -30-50 nm in height, with approximately eight-fold rotational
symmetry about the transport axis, an outer diameter of -100 nm, a central
transport channel -40 nm in diameter, and attached cytoplasmic and
nucleoplasmic filaments (Alber et al., 2007b; Beck et al., 2007; Stoffler et al.,
2003). The NPC is a modular structure composed of multiple copies of -30
proteins (nucleoporins, or Nups) arranged into distinct subcomplexes (Brohawn
et al., 2009; Rout et al., 2000; Schwartz, 2005). It is also dynamic, with
components possessing widely ranging resident times (Dultz et al., 2008; Rabut
109
et al., 2004). The most stable nucleoporins form the structural scaffold and are
largely organized in the heteromeric Nic96 subcomplex and the heptameric Y
complex. The scaffold connects to the nuclear envelope through interaction with
transmembrane nucleoporins; it anchors phenylalanine-glycine repeat-containing
nucleoporins that form an extended meshwork projecting into the central pore
channel that constitutes the main transport barrier (Frey and Gorlich, 2007; Rout
et al., 2003).
Considering its central role in transport and other cellular processes, a highresolution structure of the NPC is much sought after. Significant effort has
focused on elucidating the central scaffold architecture. Its fundamental
importance is evident, as assembly defects and transport deficiencies result from
knockout or knockdown of scaffold components (Boehmer et al., 2003; Fabre
and Hurt, 1997; Galy et al., 2003; Harel et al., 2003; Makio et al., 2009; Walther
et al., 2003). Still, the arrangement of the Nic96 subcomplex (including Nic96,
Nup53, Nup59, Nupl57, Nupl70, Nupl88 and Nupl92; nomenclature is from
Saccharomyces cerevisiae unless noted) remains largely enigmatic. However,
the structures of major portions of Nic96, Nup170 and Nup53/59 are now
available and combined with functional data will help narrow down the problem
(Handa et al., 2006; Jeudy and Schwartz, 2007; Onischenko et al., 2009;
Schrader et al., 2008b; Whittle and Schwartz, 2009). Organization of the Y
complex (composed of Nup133, Nup84, Nup145C, Sec13, Nup120, Nup85 and
Seh1) is better understood. The complex is tightly associated and forms a Y, as
observed by EM, with two short arms and a long kinked arm connected at a
central hub (Harel et al., 2003; Kampmann and Blobel, 2009; Lutzmann et al.,
2002; Siniossoglou et al., 2000; Walther et al., 2003). High-affinity connections
within the Y complex involve binary interactions between a-helical domains of its
constituents (Boehmer et al., 2008; Brohawn et al., 2008; Lutzmann et al., 2002).
The long arm terminates with a flexibly attached N-terminal p-propeller of
Nup1 33 followed by an irregular a-helical stack domain that interacts end-to-end
with Nup84 (Berke et al., 2004; Boehmer et al., 2008; Whittle and Schwartz,
110
2009). Nup84 in turn interacts with Nupl45C. Nupl20 and Nup85 form the short
arms of the Y complex, with the C-terminal region of Nupl20 forming the central
hub that interacts with the C-terminal tail modules of Nupl45C and Nup85 (Leksa
et al., 2009). Nupl45C and Nup85 bind the related p-propeller proteins Sec13
and Seh1, respectively, by addition of an N-terminal insertion blade to complete
the open six-bladed p-propellers in trans (Brohawn et al., 2008; Debler et al.,
2008; Hsia et al., 2007). Although crystallographic data on single proteins and
some binary complexes are available, we are still lacking a detailed structural
description of the entire Y complex, notably including all domain-domain and
protein-protein interfaces.
A common evolutionary origin of the NPC and vesicle coats has been proposed
based on their shared role in stabilizing curved membranes and predicted
similarities in fold composition of constituent proteins (Devos et al., 2004).
Structural evidence of a common ancestor has been obtained, as the
nucleoporins Nic96, Nup85, Nup145C and Nup84 are homologous to the COPII
vesicle coatomer Sec3l and together constitute the unique fold class ACE1
(ancestral coatomer element 1; (Brohawn et al., 2008)). In the COPII vesicle coat
lattice, two molecules of the ACE1 protein Sec3l interact to form edge elements,
whereas the p-propellers of Sec3l and Sec1 3 interact to form vertex elements
(Fath et al., 2007; Stagg et al., 2008). We proposed that the NPC structural
scaffold forms a similar lattice-like coat for the nuclear envelope (Brohawn et al.,
2008; Brohawn and Schwartz, 2009a). However, the absence of structural
knowledge of ACE1 organization in the NPC precluded any direct comparison of
the NPC and COPIl lattices.
We set out to determine the molecular architecture of an edge element in the
NPC lattice and here present the 4.0-A crystal structure of the heterotrimeric
Nup84-Nupl45C*Secl3 unit of the Y complex of the NPC from S. cerevisiae.
The ACE1 interaction between Nup84 and Nup145C is architecturally related to
the Sec3l edge element in the COPII lattice. As in the COPIl coat, the edge
111
element in the NPC lattice is arranged in a manner consistent with its role in
stabilizing membrane curvature at the nuclear envelope. We further present a
composite atomic model of the Y complex, propose how it is arranged in the
NPC, and compare the NPC lattice architecture to that of vesicle coats.
Results
Structure of Nup84-Nup145C*Sec13 trimeric complex
We solved the crystal structure of a trimeric complex between Nup84, Nup145C,
and Sec1 3 from S. cerevisiae by multiple isomorphous replacement with
anomalous scattering using selenomethionine and tantalum bromide derivatives
(Figure 4.1 and Table 4.1). The crystallized construct includes Nup84 1- 24 ,
Nup145C 10o. 555 and full-length Secl3. Despite modest resolution and relatively
high B factors, initial phase estimates were excellent and resulted in high-quality
electron density maps (Figure 4.2). Still, confident model building at this
resolution is aided by the availability of high-resolution models of fragments of
the structure (Brunger et al., 2009). To this end, we crystallized a complex of the
minimal interaction domain of Nup145C 109_179 in complex with full-length Secl3,
solved the structure by molecular replacement at 2.6-A resolution, and refined it
to an Rwork/Rfree of 21.5%/25.3%. We placed this partial model and Nup145C18o_
5ss from the S. cerevisiae-H. sapiens hybrid Nup145C-Sec13 structure (Hsia et
al., 2007) into the map using real-space methods. Phase improvement using the
experimental data with the partial model resulted in clearly interpretable maps
into which we built the final Nup84-Nup145C-Sec13 model and refined it to an
Rwork/Rfree of 28.2%/32.9%.
112
Table 4.1 - Data collection and refinement statistics
Nup145C 10 s.179-Sec13
Native
Nup84-Nup145C-Sec13
Selenomethionine
Nup84-Nup145C-Sec13
[Ta6 Br12]2 +
Data collection
Space group
Cell dimensions
a, b, c (A)
P21212
P6222
P6222
68.3, 93.9, 55.0
170, 170, 271
170, 170, 270
a, #, y (0)
90, 90, 90
90, 90, 120
90, 90, 120
50-4.0 (4.14-4.0)
50-4.4 (4.56-4.4)
Resolution (A)
40-2.6 (2.66-2.6)
*
Rsym(%)
/ /al
12.6 (64.9)
12.4(2.0)
17.2 (97.4)
10.6(1.5)
17.0 (79.9)
8.6(1.3)
Completeness (%)
Redundancy
97.9 (96.9)
3.5 (3.3)
99.9 (99.9)
5.9 (5.0)
92.2 (76.6)
3.7 (2.2)
35-2.6
50-4.0
Refinement
Resolution (A)
No. reflections
Rwork / Rree
No. atoms
Protein
Water
B-factors (A2)
Protein
Water
R.m.s deviations
Bond lengths (A)
Bond angles (0)
11148
37016
21.7/25.4
28.2/32.9
2621
67
8671
0
71
70
189
n/a
0.003
0.004
0.679
0.762
*Values in parentheses are for highest-resolution shell.
113
A.
B.
Nup84
Nup145C
N Wnrmnus
C.
Ntrmnus
Cterminus
114
Figure 4.1 - Structure of Nup84*Nup145C*Sec13
(A) Y complex of the NPC. The crystallized trimeric segment is colored. (B)
Overall structure of the heterotrimeric Nup84eNupl45C*Secl3 complex, with
Nup84 in green, Nupl45C in blue, and Secl3 in light orange. P-propeller
composed of blades 1-6 from Secl3 and blade 7 from Nupl45C is labeled. (C)
Structure is rotated by 90*, with secondary structure elements of Nup84 and
Nup145C labeled.
Nup84
Nupl45C
Secl3
Figure 4.2 - Electron density for the Nup84.Nup145C-Sec13 model
Phase combined 2Fo-Fo 4.0 A electron density map contoured at 1.5 a, shown in
gray. Heavy atom electron density displayed for the 12 selenium sites (red, 8 a)
and 4 tantalum sites (black, 6 a). The structure is shown as a Ca trace.
115
The trimeric complex has the approximate shape of a kinked rod, with
dimensions -150 x 30 x 30 A (Figure 4.1). Nup84 and Nupl45C both form ahelical blocks with dimensions -65 x 30 x 30 A that interact at the kink in the rod,
creating a 2,040-A 2 interface. The N terminus of Nup145C forms an insertion
blade that completes the open six-bladed p-propeller of Sec1 3 in trans. The
higher-resolution fragments both superimpose well with the same regions in the
trimeric structure, with mostly minor deviations observed (see Methods). In
comparison to the hybrid human Secl3-yeast Nup145C structure (Hsia et al.,
2007), there is a ~10* rotation of the propeller unit about its central axis. This
may be indicative of flexibility of the p-propeller unit relative to the ACE1 domain,
which could be important in the assembly of the NPC lattice (Stagg et al., 2008)
(see below).
ACE1 nucleoporins Nup84 and Nup145C interact crown to crown
As predicted by structural modeling, Nup84 adopts an ACE1 fold despite very
low sequence homology to other ACE1 members (Brohawn et al., 2008) (Figure
4.3). ACE1 is a tripartite, J-shaped helical fold composed of three modules:
crown, trunk, and tail.
The Nup84*Nupl45C*Secl3 structure contains the trunk and crown modules of
Nup145C and Nup84 (Figure 4.3). The two proteins form an extensive interface
between their crown modules, with a6, a7 and a8 of Nup84 packing antiparallel
to a6, a7 and a8 of Nup145C, completely burying a7 from each protein in the
interface (Figure 4.4). The surfaces of helices a6, a7 and a8 in each protein are
distinctly hydrophobic and highly conserved (Figure 4.5). Homodimerization of
Sec3l is similarly accomplished by the antiparallel interaction of helices a6, a7
and a8 ((Fath et al., 2007); Figure 4.4). Notably, a domain swap between crown
helices a5, a6 and a7 from each Sec3l monomer is observed in the crystal
structure. Whether this is the physiologically relevant manner of interaction is
116
unclear, but a long loop that allows for the domain swap is conserved in length in
Sec3l. Regardless, the interaction likewise juxtaposes and buries a7 from each
Sec3l molecule.
117
|
ACE1
|I
| Nup145C |I
INUp8
L
It
I(%
Figure 4.3 - Nup84 is an ACEI -containing protein
Structural alignment of the canonical ACE1 fold, Nup145C, and Nup84. The 28
conserved ACE1 helices are colored in a blue-white gradient from N to C
terminus, protein-specific elaborations in Nup145C and Nup84 are in yellow.
Nupl45C contains an additional N-terminal helix, the Sec1 3-interacting insertion
blade, and a p-hairpin following al, Nup84 contains a few short 1-3 turn a-helices
following a3, a4, and a7. The canonical ACE1 structure was generated for
illustrative purposes by superimposing the crown, trunk, and tail modules of
ACE1 structures individually and constructing the conserved helices and
intervening loops in the most frequently observed position. The
Nup84-Nupl45C-Secl3 structure includes the crown and trunk modules of
Nup145C and Nup84. The characteristic a-helical architecture of the ACE1 fold is
clearly observed in Nup145C and Nup84.
118
Nup145C
Sec31
Nup84
Sec31'
Figure 4.4 - The crown-crown interaction of Nup84*Nupl45C is analogous
to that of Sec3l *Sec3l
(A) ACE1 crown-crown interaction between Nup145C and Nup84. (B) The same
interaction between two Sec3l molecules (PDB 2PM6; (Fath et al., 2007)). The
Sec3l interaction is shown without domain swapping, and the rest of the
molecules are removed for clarity (see Results). Analogous juxtaposition of
crown helices a6, a7 and a8 is observed in both (A) and (B).
119
Nup84
Nupl45C
A.
~cot
B.
C.
a6
a7
AS
OJNMl
Q~DY!LG&
4
E
AM
PD
.V.C
;orYIW
NTOO
VTN' M9CIANEPV8PTIC
L
;
a6
o8
1jl
GCVR4 0Y1
1
LIAC
L
TM Cf7"7T-V''-il
W -RA
Vpre
TNA
0
a7
as
IVCOTUTVW'u
E~fOUM
AO~I8
Figure 4.5 - The hydrophobic and highly conserved Nup84*Nup145C
interface
The Nup84-Nup145C interface is shown in an "open-book" view relative to Figure
5.1C. In (A), the surface buried upon interaction is yellow and contributing helices
and residues from each protein are labeled. Residues mutated on the surface of
helix a7 in each protein that eliminate binding in vitro and in vivo (Brohawn et al.,
2008) are labeled in red. The interface is highly complementary and largely
hydrophobic. In (B), the buried surface of each protein is outlined in black and the
protein surface is colored by conservation from white (not conserved) to orange
120
1I
W
(highly conserved). Surface corresponding to the residues mutated to abolish
binding is indicated by an inner black outline. A sequence alignment from
budding yeast for each protein from a6-a8 is shown in (C)and colored as the
surface in (B). A plot of buried surface area vs. residue is shown as a bar graph
above the alignment. The mutations made are indicated above the appropriate
residues (1206D/M21OD in Nup84 and V321E/S324E/Y325A in Nupl45C). The
mutated residues are all among the largest contributors to buried surface area
and most conserved in the interface.
Unique features of each ACE1 unit in the Nup84-Nup145C-Sec13 structure form
additional interaction sites that frame the primary surface of a6, a7 and a8
(Figure 4.5). Two extended and conserved loops in Nup84 (between a3 and a4,
and between a7 and a8) pack against the long and kinked helix a4 of Nup145C.
On the opposite side of the a6, a7 and a8 interface, Nup84 has an insertion of
three short helices between ACE1 helices a4 and a5 (a4a, a4b and a4c) that
together form an interface with two crown loops (between a6 and a7, and
between a8 and a9) of Nup145C.
Structural evidence for lattice model of NPC
By analogy to the Sec3l interaction in COPII coats, Nup84 and Nup145C have
been predicted to interact through their crown modules (Brohawn et al., 2008).
Point mutation resulting in surface residue alterations in Nup145C a7 (V321E,
S324E and Y325A) and corresponding alterations in the then-predicted Nup84
helix a7 (1206D and M210D) abrogate high-affinity binding. The structure
presented here definitively shows that the interaction between Nup84 and
Nup145C occurs through ACE1 crown modules and allows the mutant data to be
explained from a structural perspective. The altered sites on each protein are
intimately involved in the interaction surface: 1206 and M21 0 account for 11 % of
the total area of Nup84 buried (223 of 2,024 A2), whereas V321, S324 and Y325
form 12% of the total area of Nup145C buried (257 of 2,059 A2 ). Introduction of
charged residues into, or loss of large side chains from, the hydrophobic and
complementary interaction surface is highly destabilizing, resulting in specific
disruption of the interaction. That these point mutations eliminate binding
indicates that the a6, a7 and a8 surface is the primary binding determinant, and
121
the secondary framing interactions are insufficient to independently maintain
interaction.
The Nup84*Nupl45C*Secl3 structure presented here fully supports our lattice
model for the NPC and provides conclusive evidence against the alternative
'fence-like' model, based primarily on crystal contacts observed in the yeast
Nup145C*human Sec13 hybrid structure (Hsia et al., 2007). In that crystal,
Nup145C-Sec13 units stack by homotypic crown-crown interaction of Nup145C.
Superposition of the Nup145C interaction observed in that crystal with the
Nup84eNup145C interface reported here shows that formation of the two
interfaces is mutually exclusive (Figure 4.6).
122
Secl3'
Sec13
Sec13
Nup84-Nup145C-Sec13
NPC edge element
Nupi 45C-Secl 3/ Nup1 45C'-Secl 3'
Crystal packing Interaction
Figure 4.6 - The Nup84-Nupl45C-Secl3 edge element sterically clashes
with a proposed "fence-like" pole in the NPC
(A) Surface representation of the entire Nup84-Nupl45C-Secl3 structure. (B)
Two heterodimers of the yeast Nupl45C-human Secl3 structure (Hsia et al.,
2007) involved in a crystal contact are shown with the lower heterodimer aligned
to (A). The view in (A,B) is rotated 90* clockwise from Figure 5.1C. The position
of Nup84 in the physiologically relevant trimeric complex (A) sterically clashes
with the position of the second Nupl45C-Secl3 dimer related by crystal packing
(B)thus providing conclusive evidence against this interaction as part of "pole" in
the "fence-like" model of the NPC (Debler et al., 2008; Hsia et al., 2007).
Comparison of edge elements in NPC and COPiI coat
The similarity between the Nup84*Nupl45C*Secl3 structure and the
Secl3oSec3l edge element in the COPII cage is immediately apparent (Figure
4.7), so we refer to the Nup84*Nupl45C unit as an edge element in the NPC
lattice. The shared binding mode between crown modules in the two structures
123
results in analogous relative orientations of the interacting ACE1 units. The
interface between Nup145C and Nup84 creates an angle of ~120* between
ACE1 units. The interface between Sec3l molecules is ~165* in the crystal
structure, though it was modeled to be ~135* by normal mode analysis for fitting
into both COPII coat EM reconstructions (Fath et al., 2007; Stagg et al., 2008). A
hinge movement about the crown-crown interface was thus postulated to be one
mechanism that allows the coat to adapt its size to vesicles of different diameters
(Fath et al., 2007). EM reconstructions of the Y complex have similarly shown
plasticity in the angles of the long arm (Kampmann and Blobel, 2009). A similar
hinge at the Nup84*Nupl45C interface could be used in rearrangements of the
NPC lattice in assembly and transport. Consistently, we observed hinge
movement at the crown-crown interface in normal mode analysis of the
Nup84*Nupl45C-Secl3 structure (data not shown).
124
Nup84-Nup1 45C
Sec31-Sec31
A.
Nup84
Nupl45C
Sec3l
Sec3l'
Cterminus
C
170.
-
N terminus C terminus
180*
90*
-90*
B.
Nup84
Nup145C
Sec3l
N
Sec31'
teminu
N terminus
C terminus,
Nterminus
N terminus
C terminus
C terminus
6*Ctriu
Figure 4.7 - Comparison of edge elements in the NPC and COPII lattices
(A,B) The lattice edge element Nup84*Nupl45C in the NPC and Sec3l *Sec3l
(PDB 2PM6 (Fath et al., 2007)) in the COPII vesicle coat are shown as cartoons
in a half-transparent surface. The two ACE1 units in each edge element are
colored by module, with trunks orange and crowns blue. Yellow lines indicate the
interface between crown modules. The structures are shown from a top view in
(A) (rotated 1800 from Figure 4.1) and a side view rotated by 90* in (B). The
analogous crown-crown interactions result in edge elements that share a
common architectural arrangement. Viewed from the top, the Nup84*Nupl45C
edge element is bent -10* from horizontal, whereas the Sec3l Sec31 edge is
essentially straight. Viewed from the side, the crystal structures of the edge
elements show markedly different angles, with the Nup84eNupl45C edge 450
more acute than that of Sec3l*Sec3l. The angle observed in the
Nup84*Nupl45C edge corresponds closely to the angle to which the
Sec3l *Sec31 interface was modeled for fitting into the EM reconstructions of the
COPII cage and coat (Fath et al., 2007; Stagg et al., 2008). The proposed
position of the nuclear envelope membrane relative to the NPC edge element
shown in (B) is analogous to the known position of the COPIl vesicle membrane
relative to the COPIl edge element.
125
The insertion blade interaction between Secl3 and Nupl45C or Sec3l is very
similar in the two structures (data not shown). However, the different orientations
of the insertion blade with respect to the ACE1 trunks result in Sec1 3 being
positioned differently with respect to the edge elements. Inthe Sec3l *Secl3
structure, the Secl3 propeller sits against the end of the Sec3l trunk, capping
the edge element. Inthe Nup84-Nup145C-Sec13 structure, Sec13 is rotated
~45* forward toward the trunk and clockwise (viewed from the vertex) and rests
on top of the Nup145C trunk. It remains to be determined whether additional
interactions of Sec13 in the context of the entire NPC scaffold result in a
conformational change from this position.
Discussion
The Nup84-Nupl45C*Secl3 structure presented here, together with the
previously reported structures of Nup85-Sehl, Nup84*Nupl33 and Nup120
(Berke et al., 2004; Boehmer et al., 2008; Leksa et al., 2009) allows for the
generation of a composite model for the majority of the Y complex at high
resolution, including relative orientations of components in the long arm (Figure
4.8). The last four trunk helices of Nup84 need to be modeled to connect the
Nup84-Nup145C-Sec13 and Nup84*Nupl33 crystal structures. As these helices
adopt identical topologies in other ACE1 structures and are predicted to be the
only secondary structure elements present in Nup84 in this region, we can model
their structure with high confidence (data not shown). This allows us to place the
tail of Nup84 interacting with the full helical region of Nupl33 relative to
Nup84*Nupl45C-Secl3. The position of the p-propeller of Nupl33 is unknown
and is probably flexible (Berke et al., 2004). The tail modules of both Nup145C
and Nup85 can be confidently modeled (Brohawn et al., 2008), but the C-terminal
interaction domain of Nup120 cannot and is not shown. Although the relative
positions of the short arms with respect to the long arm of the Y complex cannot
be assigned unambiguously, we have chosen to model the p-propellers of Sec13
and Seh1 in close proximity to one another by analogy to the interactions of ppropellers at the vertex elements in the COPII coat (Fath et al., 2007; Stagg et
126
al., 2008). Our positioning of the short arms is most consistent with all available
data, although we cannot currently exclude alternative arrangements.
Our model is generally consistent with the recently reported EM reconstruction of
the Y complex from yeast (Kampmann and Blobel, 2009). The angles of the
Nup84-Nup145C and Nup84eNup133 interfaces in our model correspond to
those found in the highest-frequency EM class average. Here we incorporate
-0.5 MDa (of 0.58 MDa) of atomic models into a composite Y complex model.
Most notably, the connecting Nup84-Nupl45C-Secl3 structure allows for the
incorporation of relative orientations of the proteins into the Y complex model.
Analysis of the Y complex model reveals a number of functionally important
implications.
Given the high degree of conservation between edge element structures in the
NPC and COPII lattices, we predict the same inner concave surface of the edge
element will face the membrane in the NPC (Figures 4.7 & 4.8). Inthis
orientation, the N-terminal p-propeller-a-helical domain of Nup120 and the Cterminal a-helical domain of Nup1 33 point toward the membrane. These domains
could serve as attachment sites for additional nucleoporins that could connect
the Y complex to the membrane-proximal and membrane-spanning nucleoporins.
Consistently, Nupl20 interacts in vitro with Nupl57, a member of the Nic96
subcomplex that can provide a link to transmembrane nucleoporins (Lutzmann et
al., 2005; Onischenko et al., 2009). The ACE1-containing Nup85 is positioned
away from the membrane, where it may form interactions to propagate the NPC
lattice. The N terminus of Nup145C is also oriented away from the membrane,
allowing its binding partner Nup145N to project its phenylalanine-glycine repeats
into the pore channel (Ratner et al., 2007).
127
...
............
.................................
.........
Figure 4.8 - Nup84*Nupl45C is a membrane curvature-stabilizing edge
element in the NPC lattice
A composite atomic model for the Y complex of the NPC, emphasizing the role of
the Nup84*Nupl45C edge element as a membrane curvature-stabilizing unit
analogous to the Sec3l *Sec3l edge element in COPII vesicle coats. The long
arm of the Y complex is a composite model from crystal structures and is shown
with Nup145C in blue, Sec13 in orange, Nup84 in green and Nup133 in yellow.
The relative position of the N-terminal propeller of Nup1 33 (yellow) and the short
arm components Nup120 (blue) and Nup85*Sehl (blue-orange) are more
tentatively placed and shown half-transparent (see Results for details). The long
128
................
PI
axis of the Y complex is oriented along the positively curved nuclear envelope
membrane, with the concave face of the Nup84*Nup145C edge element facing
the lipid bilayer. This orientation is analogous to that of the Sec3l eSec3l edge
element in the COPIl coat and is consistent with the evolutionary relationship
between the NPC and COPiI vesicle coat lattices. Notably, although the Y
complex is shown facing the membrane, it is not predicted to directly contact the
nuclear envelope. Rather, other nucleoporins are predicted to have roles that
correspond to adaptor complexes in other vesicle coating systems that link the
membrane curvature-stabilizing coat (the Y complex) to the nuclear envelope.
The branch point in the Y complex has the P-propeller proteins Secl3 and Seh1
available to generate potential vertex interactions in the NPC lattice, similar to the
Sec3l p-propeller vertex interactions in COPII coats. In contrast, at the opposite
side of the NPC edge element, Nup84 (unlike Nup145C, Nup85 and Sec3l) does
not !interact with a p-propeller. The loss of a p-propeller, combined with the
acquisition of the Nup133 'cap', might have evolved as a way to terminate lattice
propagation in this direction of the Y complex long arm. The utility of this type of
arrangement is unique to the cytoplasmic- and nucleoplasmic-facing sides of a
NPC lattice, as it cannot form self-enclosed structures observed in vesicle coats.
Our model is consistent with a role for the NPC edge element in stabilizing
membrane curvature. In other membrane coating systems, proteins that directly
contact membranes display a positively charged surface for electrostatic
interactions with membrane phospholipids (McMahon and Gallop, 2005; Shibata
et al., 2009; Zimmerberg and Kozlov, 2006). Like clathrin and Sec3l-Secl3, the
NPC edge element does not display such a surface (Figure 4.9) and is likely to
coat to stabilize, but not directly interact with, curved membranes.
To date, the NPC has been shown to be architecturally related only to COPII
coats; its relationship to clathrin coats (Fotin et al., 2004) is limited to a shared
fold composition of components (Devos et al., 2006). Notably, an ALPS motif in
human Nup133 has been shown to associate with membranes and has been
suggested to initiate membrane curvature (Drin et al., 2007), though it has not
been found in S. cerevisiae. This site is far enough removed from the ACE1 edge
129
element that the Y complex could have both roles: a curvature initiator at the
distal end of the long arm, and a lattice-integral stabilizer at the ACE1 edge
element.
We favor a model inwhich the membrane-facing edge element of the Y complex
is oriented parallel to the transport axis and serves to stabilize the positive
membrane curvature of the nuclear envelope, consistent with the evolutionary
relationship with the COPII edge element that stabilizes positive vesicle
membrane curvature. As more high-resolution structures of components are
solved, they could be integrated to generate a more precise overall NPC
structure. Fundamental to this goal will be the elucidation of potential vertex and
inter-subcomplex interactions in the NPC lattice.
130
Sec13
.-90
Figure 4.9 - The Y complex does not display a positively charged surface
characteristic of membrane interaction
The surface of a composite structure of the long arm of the Y complex
(Nupl33-Nup84-Nupl5C-Secl3) is shown colored according to electrostatic
potential from red (-7 kT/e) to blue (+7 kT/e) in four views consecutively rotated
90*. The top view corresponds to the view in Figure 4.7B. The second view is of
the concave face of the Nup84-Nup145C edge element predicted to face the
membrane by analogy to the orientation of the Sec3l -Sec3l edge element in the
COPII lattice.
131
Methods
Construct generation
We cloned the trimeric complex of Nup84 (residues 1-424), Nup145C (109-555)
and Sec1 3 from S. cerevisiae into a bicistronic bacterial expression vector.
Nup84 1-424 was N-terminally fused with a cleavable 6xHis-tag. Nup145C109.555
was C-terminally fused to Sec13 with a flexible 9-residue linker, to increase
complex stability, without affecting chromatic behavior compared to the separate
chain complex (data not shown, (Brohawn et al., 2008)). The trimeric complex is
referred to as Nup84*Nupl45C*Secl3 for simplicity. The completed p-propeller
construct of Secl3 was generated by fusing the insertion blade of Nupl45C
(residues 109-179) C-terminally to full-length Secl3 via a flexible 9-residue
linker. Secl3 was N-terminally fused with a cleavable 6xHis-tag.
Protein production and purification
Proteins were expressed in E. coli BL21-RIL(DE3) cells and purified as described
(Brohawn, Leksa et al. 2008). Eluted protein was dialyzed against 50 mM HepesNaOH pH 7.4, 200 mM NaCl, 1 mM DTT, and 0.1 mM EDTA, the tag cleaved
with protease overnight, and purified on a HiTrapS column (GE Healthcare) via a
linear NaCI gradient followed by size exclusion chromatography. Nup145C1o.
179*Sec13 was purified using a Superdex S75 26/60 column (GE Healthcare) run
in 10 mM Tris-HCI pH 8.0, 150 mM NaCl, 1 mM DTT, and 0.1 mM EDTA.
Nup84*Nupl45C-Secl3 was purified using a Superdex S200 26/60 column (GE
Healthcare) run in 10 mM Tris-HCI pH 8.0, 200 mM NaCI, 1 mM DTT, and 0.1
mM EDTA. Selenomethionine-derivatized Nup84*Nup145C-Secl3 was prepared
as described (Brohawn et al., 2008) and purified as the native version with
reducing agent concentration at 5 mM in all buffers.
Crystallization
Small crystals of Nup145C 10 9.179 *Sec13 grew in hanging drops of 0.5 pl protein at
85 mg ml- 1 and 0.5 pl precipitant (0.1 M Tris-HCI pH 8.3, 26.5% (w/v) PEG 4000,
132
0.25 M LiCI) at 160C in three days and were processed for seeds. Diffraction
quallity crystals grew as large plates (300 x 300 x 10 pm) in hanging drops of 0.2
pl seed dilution, 0.5 pl protein at 38 mg ml- 1, and 0.5 pl precipitant (0.1M Tris-HCI
pH 8.3, 22% (w/v) PEG 4000, 0.25 M LiCI) at 16*C in three days. Crystals were
cryoprotected by briefly soaking in precipitant with 25% v/v glycerol and flash
frozen in liquid nitrogen.
Diffraction quality crystals of Nup84-Nupl45C*Sec13 grew as half-cylinders 2550 pm in height with a radius of 50-200 pm in hanging drops of 0.25 pl protein at
22.5 mg ml- 1 and 0.25 pl precipitant (1.15 M sodium malonate pH 5.7) at 220C in
2 days. Selenomethionine derivatized protein crystallized under identical
conditions. [Ta6 Br12]2+-derivatized crystals were obtained by transferring crystals
into a 0.5 pl drop of 1.17 M sodium malonate pH 5.7 and 200 pM [Ta6 Br12 ]2 +X
2Br- (Jena Biosciences) and incubating for 1-2 hours. Crystals were
cryoprotected by briefly soaking in precipitant with 22.5% v/v ethylene glycol and
flash frozen in liquid nitrogen.
Data collection and structure determination
iMosflm (Leslie, 1992) was used for data collection strategies, HKL2000
(Otwinowski and Minor, 1997) was used to reduce data, and model building and
refinement were carried out with Coot (Emsley and Cowtan, 2004) and Phenix
(Adams et al., 2002).
A 20 pm aperture beam was used to collect data from separate spot regions of
the Nup145C 109.179-Secl3 crystals because diffraction quality varied over their
volume. Molecular replacement was accomplished with Phaser (McCoy et al.,
2007) using Secl3 (PDB ID 2PM6 (Fath et al., 2007)) as a search model. The
final model is missing the first two residues of Sec13, as well as loop residues
158-167. The final model has Ramachandran plot values of 95.4% favored, 4.3%
allowed, and 0.3% outliers.
133
The structure of Nup84-Nupl45C-Secl3 was solved with multiple isomorphous
replacement with anomalous scattering (MIRAS) using selenomethionine and
[Ta6Br1 2]2+derivatives. 12 selenium sites (out of 13) and 4 [Ta6Br1 2]2+sites were
found with SHELXC/D/E (Sheldrick, 2008) and refined in SHARP (Vonrhein et
al., 2007). Nup145C109 .179 *Secl3 (this work) and Nup145C 18o-555 (PDB accession
code 3BG1 (Hsia et al., 2007)) were placed into the solvent-flattened map from
SHARP with BrutePTF (Strokopytov et al., 2005). PhaserEP (McCoy et al., 2007)
was used to refine selenium sites and the partial model. Discussion refers to the
final selenomethionine crystal as it had a lower B factor and more interpretable
maps than native crystals without any appreciable differences in the overall
structure (data not shown). Residues 3-7 of Secl3, 554-555 of Nupl45C, and 132 of Nup84 are not modeled, in addition to residues missing in Nup145C1 0o.
179*Secl3.The absence of observed density for al of Nup84 may be due to the
absence of ACE1 helix a17 in the crystal construct, which typically interacts with
helix al inACE1 proteins. The final model has Ramachandran plot values of
87.9% allowed, 10.9% allowed, and 1.2% outliers.
The high-resolution structure fragments superimpose well with the corresponding
regions in the complete Nup84*Nupl45C*Secl3 structure. Nup145C1 09.179-Secl 3
aligns with the same region in the trimeric structure with an average rmsd of 0.75
A. The major difference is the orientation of the N-terminal 11 residues of Sec1 3.
Inthe Nup145C1 09- 179-Secl3 structure, this region is extended away from the
molecule and amino acids 3-8 from Secl3 form a strand E zipper closure with
strand D from blade 2 of a neighboring Sec1 3 molecule. A short strand is formed
from part of the loop connecting Secl3 and Nup145C and forms strand F. Inthe
Nup84-Nupl45C-Secl3 crystal, this interaction is not possible as there is not a
symmetry related Secl3 molecule in an equivalent position. Instead, the Nterminus of Sec13 extends towards the N-terminal helix of Nupl45C, though it is
not modeled. We presume that the zipper interaction of two Sec1 3 molecules
and the linker in the Nup145C10 ..179 *Secl3 structure is a crystal-packing artifact.
Nupl45C*Secl3 in the trimeric structure overlays well with the reported S.
134
cerevisiae / H. sapiens hybrid structure with an average rmsd of 1.2 A. The major
difference between the two structures is a rotation of the Nup145C insertion
bladle/Sec1 3 unit of ~5-10 about the propeller axis. Whether this is a relevant
movement of the molecules remains to be determined. Some rearrangement in
the crown of Nup145C is observed in the current structure that is accounted for
by reordering to form the interaction site for Nup84.
Structure analysis
Structure figures were made in Pymol (http://www.pvmol.ora). Interface
calculations were performed using the PISA server (Krissinel and Henrick, 2004).
Alignments were made with MUSCLE (Edgar, 2004), analyzed in Jalview
(Waterhouse et al., 2009), and figures produced with Aline (Bond and
Schuttelkopf, 2009). Structural superpositions were performed with Coot
(Krissinel and Henrick, 2004) and Cealign (Jia et al., 2004).
Acknowledgements
We thank the staff at beamlines 24-ID-C/-E at Argonne National Laboratory for
assistance with data collection, the staff at beamline X29 at the National
Synchrotron Light Source for assistance in screening cryoprotection conditions
through mail-in data collection service, M. Gogola for his contributions to the
Nup145C10 9. 179-Sec13 structure, J. Iwasa for help with Figure 4.8, and members
of the Schwartz laboratory for valuable discussions. This work was supported by
NIH grant GM77537 (T.U.S.), a Pew Scholar Award (T.U.S.), and a Koch
Fellowship Award (S.G.B.)
135
Chapter five
Extending the lattice model of the NPC
136
A remarkable amount of insight into the structure and evolutionary origins of the
nuclear pore complex (NPC) has been gained in the past several years. Though
a relatively small percentage of the NPC has been structurally characterized in
that time, it has provided us not only with firm evidence of a common ancestry of
the NPC and COPII vesicle coats by virtue of the shared ancestral coatomer
element 1 (ACE1) fold, but a means to create a conceptual framework for the
entire NPC structure. Here I discuss future directions of research into the
structure of the NPC, expand upon potential parallels to other vesicle coating
systems, and hypothesize how other components of the NPC could be
incorporated into the assembly based on our working model of the NPC lattice.
From A to Y: a complete Y complex structure
The most direct extension of work described in this thesis is the complete
structural characterization of the Y complex. To date, we have x-ray structures
for -500 kDa of the 575 kDa complex (-87%) combined from yeast and human
(Berke et al., 2004; Boehmer et al., 2008; Brohawn et al., 2008; Brohawn and
Schwartz, 2009b; Leksa et al., 2009; Whittle and Schwartz, 2009). What remains
is a short 4-helix stretch between the ACE1 trunk and tail of Nup84 and the "hub"
of the Y complex formed by the interaction of the C-terminal (-35 kDa) domain of
Nup120 with the tail modules Nup145C and Nup85 (-20 kDa each).
The portion of Nup84 that is structurally uncharacterized can be confidently
predicted to be a group of 4 ACE1 helices that form the transition from trunk to
tail modules. This region bridges the structures of the S. cerevisiae Nup84 trunk
and crown modules and the H. sapiens Nup107 (Nup84 homolog) tail module. It
is clear from proteolysis experiments that the region is flexible in H. sapiens
Nup107 (Boehmer et al., 2008), and therefore is likely to be difficult to crystallize.
Several constructs of S. cerevisiae Nup84-Nupl45C*Secl3 that include the
missing section were made and evaluated, but did not crystallize in initial
screening attempts. Perhaps the most likely to succeed approach would be to
design S. cerevisiae Nup84 tail constructs in complex with Nup133 by analogy to
137
the Nup107-Nup133 structure that include the missing region in Nup84. If
successful, this would have the additional benefit of providing structural
information for S. cerevisiae Nup133, which we currently model based on the H.
sapiens structure. Another approach would be to design constructs of Nup84 that
splice out the crown module and contain just tail and trunk. While the structure of
this region will show the orientation of the Nup84 modules with respect to one
another, it is almost certain to verify our predicted model (Brohawn and
Schwartz, 2009b) and is thus somewhat of a lower priority for future efforts.
In contrast, the uncharacterized hub of the Y complex is an immediately
important next structural target. The hub structure will likely prove to be important
for several reasons. First, it is still unclear from bioinformatic prediction methods
what structure the C-terminal domain of Nup120 will adopt, apart form it being
predominantly helical. Second, while we can confidently predict the overall fold of
the tail modules of Nup145C and Nup85 (Brohawn et al., 2008), the manner in
which they will interact with Nup120 is unclear. The interaction could be arranged
similarly to that between the ACE1 tail module of Nup84 and Nup133 (Boehmer
et al., 2008) in which the terminal helices of Nup84 bind Nup1 33. Consistently,
we have shown that the C-terminal helix of Nup145C is involved in the interaction
with Nup120 (Figure 2.7). However, Nup120 and Nup133 do not appear to be
related proteins: we have shown that their N-terminal regions adopt distinct folds
(Berke et al., 2004; Boehmer et al., 2008; Leksa et al., 2009; Whittle and
Schwartz, 2009) and their C-terminal domains do not have convincing sequence
homology. Still, this does not exclude the possibility of Nup120 adopting a similar
fold to the Nup84 binding site of Nup133 in its C-terminal region, perhaps
arranged as a tandem array in order to provide binding sites to both Nup85 and
Nup145C. Third, the structure of the hub would also answer the remaining
questions about the relative orientations of the proteins in the Y complex. We
have speculated that the P-propellers of Sec13 and Seh1 could be in close
proximity to one another in the Y complex, potentially serving as a vertex element
in the higher order assembly of the NPC (Chapter 4). Incontrast, a model
138
produced by fitting crystal structures into an EM reconstruction of the Y complex
showed the reverse orientation for Nup85-Seh1 (Kampmann and Blobel, 2009).
The structure of the hub is certain to resolve this discrepancy.
Beyond the Y: incorporation of other nucleoporins into the NPC lattice
Insights gained from structures of the majority of the Y complex and pieces of the
Nic96 complex have led us to propose a lattice model for the NPC. Of course, at
this point the model is far from complete and raises a number of questions to be
answered in the future. The most exciting lines of inquiry will extend from this
preliminary model and build up to the higher order structure of the NPC. Cleary,
the general approach taken to study the Y complex will be applied to the
remainder of the NPC. Dissection of the Nic96 complex, the Ndcl membranespanning complex, and the peripheral complexes structures can likely be
achieved in much the same manner as for the Y complex. However, insight
gained from our work to date can also be used to ask more targeted biological
questions in route to a complete atomic level description of the NPC. Specifically,
one can ask whether other Nups will have functions similar to vesicle-associated
proteins such as those that form lattice vertices or act as adaptors or tethers.
Below, hypotheses for the manner of integration and function of scaffold
nucleoporins in the NPC lattice are discussed.
p-propellers and vertex elements in the NPC lattice
One prediction from our lattice model for the NPC is that vertex elements will be
used to connect the edge elements we have identified in the Y complex.
Identifying the nucleoporins involved in putative vertex elements in the NPC
lattice and the manner of their interaction is thus a major goal of future research.
The vertex arrangement in the NPC and COPIl lattices might prove to be similar.
The presence of Sec13 in both the NPC and COPIl coats lends circumstantial
support to this idea. Inthe COPIl coat, 4 edge elements interact to form a vertex.
Each edge contributes two interacting p-propellers, a proximal Sec3l 7-bladed
139
P-
propeller and a distal Secl3 6-bladed P-propeller (Chapter 2, (Fath et al., 2007;
Stagg et al., 2008)). Four interactions between the P-propellers seem responsible
for forming the vertex: two are invariable contacts (one between two Sec3l
propellers and one between Secl3 and Sec3l) and two are variable (both
between neighboring Sec3l propellers) (Stagg et al., 2008). In the NPC, one
molecule of Secl3 per edge element could be used in a vertex, but what
additional propellers could be involved? Seh1 seems likely to be for several
reasons. First, it is highly homologous to Secl3 and like Secl3 it interacts with
an ACE1 protein, Nup85. Second, it is probably in close proximity to Sec1 3 in the
Y complex (Chapter 4). Lastly, recent evidence suggests it may interact with
other proteins and play a role in a separate coating complex away from the NPC.
Neither Nup145C nor Nup85 have a 7-bladed p-propeller like Sec3l, so if an
additional propeller is used in a NPC vertex it would have to be contributed from
another Nup. Candidates include the other conserved p-propellers in the Y
complex (in Nup120 or Nupl33) or the Nic96 complex (Nupl57/Nupl7O). Three
additional p-propellers interact with the Y complex in metazoans (Nup37, Nup43,
and ELYS) and though these have yet to be identified in S. cerevisiae, two are
present in A. nidulans (Nup37 and ELYS) (Brohawn et al., 2009). The functions
of these propellers have yet to be investigated.
While it seems reasonable to expect that other p-propellers in the NPC will play a
role in vertex interactions, evidence of propeller-propeller interactions is currently
lacking. This is perhaps indicative of weak interactions at the expected vertices,
which would thus not have been likely to be indentified in studies probing direct
interactions of Nups to date. Consistent with this notion is the fact that vertex
interactions in the COPIl lattice have only been characterized in coat
reconstructions by EM (Fath et al., 2007; Stagg et al., 2008). With the
expectation that vertex contacts in the NPC will be of a weak and/or transient
nature, future studies can be designed to specifically probe for these types of
interactions. It may be important to not only test interactions between known ppropellers of the NPC, but to revisit global approaches to ascertain whether
140
another p-propeller-containing protein is involved that had been missed in
previous NPC inventorying experiments.
What might be the role of p-propellers in the NPC be if they are not used in
COPII-type vertex interactions? One possibility is that some may have roles
similar to that demonstrated for the p-propeller in clathrin coats. The sevenbladed P-propeller at the N-terminus of the clathrin heavy chain is not involved in
lattice vertex interactions, but rather projects inward toward the vesicle
membrane (Fotin et al., 2004). The p-propeller binds to a wide variety of cargo
adaptors and membrane-interacting proteins and thus serves to bridge the outer
coat to the membrane and contents of the vesicle (Owen et al., 2004; Young,
2007). This is accomplished through multiple protein binding sites on the clathrin
p-propeller, present both on the top face of the propeller and in grooves on the
outer face between p-blades (ter Haar et al., 2000). One candidate p-propeller in
the |NPC for a similar role is in Nup120. In our current model for the arrangement
of the Y complex in the NPC, Nup120 extends its N-terminal p-propeller towards
the nuclear envelope (NE) membrane (Chapter 4). Inthis position, it is ideally
situated to bind to other nucleoporins that would link the outer edge element in
the Y complex to the NE. Interestingly, the p-propeller surface of Nup120
displays two regions of high sequence conservation: one on the top surface and
one on the outer face between blades 3 and 4 (Chapter 3). Nupl20 has been
shown to interact with Nup157 (Lutzmann et al., 2005), which could play a role in
linking the Y complex to the membrane (see below). Future work taking a
targeted approach to testing this interaction as well as identifying other potential
Nup120 p-propeller interacting Nups should prove interesting.
p-propellers on the periphery of the NPC are likely to be involved in scaffolding
and recruiting other proteins with functional roles at the NPC. This has been
shown to be the case for the interaction of the Nup214 p-propeller with the RNA
helicase Ddx19 at the cytoplasmic face of the NPC (Napetschnig et al., 2007;
von Moeller et al., 2009) any may also be true for Nup82 (Xu and Powers, 2009).
141
a-helical nucleoporins in the NPC lattice
a-helical domains make up -50% of the mass of the NPC (Chapter 1). Early
bioinformatic approaches predicted that all of the non coiled-coil helical domains
would adopt regular a-solenoid like folds (Devos et al., 2006). Surprisingly, each
helical domain to be structurally characterized has so far been found to fall into
one of three distinct, non a-solenoid folds: ACE1, the Nupl33/Nupl70 fold, or the
Nup120 fold (Brohawn et al., 2008; Brohawn and Schwartz, 2009b; Leksa et al.,
2009; Whittle and Schwartz, 2009). The role that the Nup120 helical domain
serves in the NPC lattice in bridging Nup145C and Nup85 in the Y complex was
discussed above. Possible functions of the ACE1 proteins not known to form
edge elements and of the Nupl33/Nupl70 fold in the lattice are discussed below.
It is sill not clear whether the two largest helical Nups, Nup1 88 and Nup1 92, will
adopt a more regular helical repeat arrangement, one of the three helical folds
seen to date in the NPC, or present yet another surprise.
ACE1 nucleoporins
Four nucleoporins (Nic96, Nup85, Nup145C, and Nup84) have been shown to
adopt ACE1 folds. As discussed previously (Chapter 4), Nup145C and Nup84
are proposed to form an edge element in the NPC lattice. However, it remains to
be determined what exactly the roles of the Nic96 and Nup85 ACEls in the NPC
lattice are. It is tempting to speculate that the ACE1 crown modules of Nic96 and
Nup85 will be involved in crown-crown interactions either homo- or heterotypically to form additional edges. However, the crowns of Nic96 and Nup85 are
distinct from other ACEls in that their surface is neither noticeably hydrophobic
nor well conserved (Brohawn et al., 2008; Jeudy and Schwartz, 2007). This
suggests that if they do participate in crown-crown interactions, they might be
weaker in nature than the edge interactions so far observed. It also is unclear
what the role of the tail module of Nic96 is, though it was predicted to form an
interaction site from structural analysis before it was recognized as an ACE1
(Jeudy and Schwartz, 2007). Structural homology between Nup1 33 (which
interacts with the Nup84 ACE1 tail) and Nupl57/Nupl70 (see below) suggest
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Nup157/Nupl70 could interact with the Nic96 tail, though this has yet to be
observed.
It may be that Nic96 and Nup85 are used differently in the NPC lattice than the
other known ACE1 proteins. Interestingly, in both crystals, pairs of molecules
form antiparallel homotypic interactions along their long axes. Nic96 has an
especially striking surface charge distribution that results in a dipole moment due
to a generally positively charged crown and negatively charged tail that may
contribute to the interaction seen in the crystal (Jeudy and Schwartz, 2007;
Schrader et al., 2008b). While these interactions are not observed in solution,
they may be relevant within the confines of the assembled NPC, with its
correspondingly high protein concentrations. Future work should elucidate the
role of these two ACE1 proteins in the NPC lattice.
Nup133/Nup17O and other helical nucleoporins - adaptors/tethers in the
NPC?
A second a-helical fold indentified in Nups is shared between the Y complex
member Nup133 and the Nic96 complex components Nup157/Nup17O (Whittle
and Schwartz, 2009). The architecture of this second ancestral element is quite
distinct from either ACE1 or regular a-solenoid like folds. While the relationship
between Nupl33 and Nupl57/Nupl70 is more distant than between ACE1s, the
proteins clearly share a stretched, multipartite helical arrangement with
conserved topology. The role for these Nups in the NPC architecture is still
uncliear, but the presence of a membrane binding ALPS motif and Nup84
interaction site in human Nup133 and evidence for Nup157/Nup17O being linked
to transmembrane Nups (possibly through Nup53/Nup59), Nup120, and
anchoring other Nups to the NPC suggests they may both act as connectors in
the lattice (Drin et al., 2007; Flemming et al., 2009; Lutzmann et al., 2005; Makio
et al., 2009; Onischenko et al., 2009).
143
A recent structure of the Ds1l vesicle-tethering complex has provided a basis for
speculation that the Nup1 33/170 fold is related to tethering complex proteins and
may play a functionally similar role in the NPC (Ren et al., 2009). Vesicle
tethering complexes are multiprotein assemblies that bridge vesicles to target
membranes, typically over large distances. The trimeric Ds11 complex is involved
in tethering COPI vesicles to the ER membrane (Sztul and Lupashin, 2009).
Vesicle interaction is accomplished by one subunit (Dsll) binding to COPI
coatomers with an extended loop. Ds11 also binds to the other two components,
Sec39 and Tip20, which are in turn anchored to the ER membrane via interaction
with ER associated SNAREs (Ren et al., 2009). Interestingly, while Sec39 of the
Dsl1 complex was described as a novel fold, its C-terminal region is reminiscent
of the Nup133/170 domain. Preliminary structural superpositions show that
Sec39 and Nupl33/170 indeed share the same fold topology in this domain.
Despite the many differences in the systems, at a simplistic level Nup133,
Nup157/Nup170, and Sec39 may have a common function in linking membranecoating proteins to membrane-binding proteins. It is thus not unreasonable to
hypothesize they evolved from an ancestor with that same capability. Whether
this relationship is meaningful or not remains to be addressed in future work.
Regardless, it seems likely that at least some nucleoporins will play an adaptorlike role in connecting the peripheral lattice of the NPC including the edge
element in the Y complex to the pore membrane. One or more Nups of the Nic96
complex (including Nup53/Nup59, Nup1 88, and Nupl92) seem most likely to
play such a role. It is possible that these adaptor interactions will be more
complex and difficult to elucidate than the strong binary interactions observed in
the Y complex. As has been seen in vesicle coat adaptor complexes, they may
involve tertiary or higher order interactions, be relatively weak or transient, or
might involve unstructured regions of peptide outside of well-ordered domains
(Edeling et al., 2006; McMahon and Mills, 2004).
144
Overall features
As structural knowledge of the NPC architecture progresses, we are able to
begin to ask questions relating to the overall NPC structure. While many issues
are likely to be resolved in the coming years, two in particular that may be
addressed by extension of the lattice model are elaborated on below.
Flexibility of the NPC
It will be interesting to see if a lattice model is able to account for structural
plasticity and deviations from eightfold symmetry within NPCs. Deviations from
eight fold symmetric NPCs have been consistently observed (Akey, 1995; Beck
et al., 2007; Frenkiel-Krispin et al., 2009) and in fact extend to the presence of
supernumerary complexes of 9 or 10 scaffold segments (Hinshaw and Milligan,
2003). Construction of the NPC as a lattice akin to vesicle coatomers suggests a
possible mechanism for these observations. In both COPII and clathrin lattices,
significant heterogeneity in coat architecture is observed, though the lattices
accomplish flexibility of assembly in fundamentally different ways. In clathrin
coated vesicles, a variety of lattice organizations are made possible by restriction
of the hub (or vertex) structure while allowing for variable interactions along the
legs (edges) of the clathrin triskelion (Fotin et al., 2004). In COPII lattices,
variation of vertex interactions has been shown to be able to account for different
coat geometries, while the edge interactions remain relatively fixed (Stagg et al.,
2008). In both cases, fixing the geometry of the edge or vertex interaction and
allowing for variability inthe other results in flexibility in lattice assembly. Once it
is determined how NPC vertex interactions are arranged, the question of whether
this principle is recapitulated in the NPC could be addressed.
Lattice architecture and membrane curvature
The nuclear pore membrane is unique in that it defined by both positive and
negative curvature. Highly curved membranes are generally unstable and so
presumably there will be mechanisms to generate and stabilize both positive and
negative membrane curvature in the pore membrane (McMahon and Gallop,
145
2005; Shibata et al., 2009; Zimmerberg and Kozlov, 2006). While we are
beginning to develop a model for membrane stabilization in the NPC, major
questions still to be resolved include how pore membrane curvature is generated
and how the negative membrane curvature is stabilized.
Inthe direction parallel to the transport axis, the pore membrane displays positive
curvature. In the COPII lattice, the Sec3l *Sec31 edge element is arranged
parallel to and stabilizes the positive curvature of the vesicle membrane (Fath et
al., 2007; Stagg et al., 2008). Based on the equivalent architecture of the edge
element in the Y complex of the NPC, we propose it will likewise be arranged
parallel to the positive membrane curvature of the pore membrane and will
function similarly (Chapter 4). A recent cryo-ET reconstruction of Xenopus NPCs
has defined densities arranged parallel to the transport axis that are roughly the
expected size of two Y complexes (Frenkiel-Krispin et al., 2009). Although it has
not yet been possible to fit high resolution models of the Y complex into the
tomographic reconstructions, these data are consistent with our model of Y
complexes arranged as struts in the NPC lattice. The surface of the Y complex
proposed to face towards the membrane is not observed to display a noticeable
positively charged surface or other characteristic consistent with direct
membrane binding (Figure 4.9). Thus, it (like the COPIl outer coat) is proposed to
play a membrane scaffolding and curvature-organizing role.
Interaction between proteins and lipid bilayers can generate curvature in different
ways that may be important in the NPC (McMahon and Gallop, 2005; Shibata et
al., 2009; Zimmerberg and Kozlov, 2006). Amphipathic helix insertion into
membrane leaflets is one mechanism that produces local regions of positive
curvature. For instance, in COPI, COPII, and clathrin-coated vesicles, positive
curvature is initiated by helix insertion of the GTPases Arf1, Sari, and dynamin,
respectively (Pucadyil and Schmid, 2009). Inthe NPC, the presence of an
amphipathic, membrane-inserting helix ('ALPS'-motif) in human Nup133 has
been demonstrated to sense positive curvature in vitro (Drin et al., 2007), though
146
the importance of this domain in vivo and its generality across species in still
unclear. Recent studies have also implicated membrane insertion by reticulons in
NPC assembly (Dawson et al., 2009). Integral membrane proteins of the NPC
might directly induce membrane curvature if the transmembrane domains are
funnel shaped and add surface area preferentially to one leaflet. Alternatively,
oligomerization of either the NE luminal or NPC facing domains of
transmembrane Nups could impart and stabilize curvature. In fact, the NE luminal
domains of the yeast transmembrane Nups have been suggested to oligomerize
into a ring structure (Alber et al., 2007a). This type of structure could be
responsible for imparting and stabilizing the negative curvature of the pore
membrane in the plane of the nuclear envelope.
Proteins that bind to the surface of lipid bilayers can also generate membrane
shape. This manner of interaction is typified by the BAR domain. BAR domains
share the characteristics of forming crescent shaped helical assemblies that bind
membranes and impart curvature. Variations include canonical BAR domains
that bind membrane surfaces with their concave face and induce positive
curvature, N-BAR domains that have an additional amphipathic helices that insert
into the membrane to further effect curvature, and inverse-BAR domains that
bind lipids with their convex face and induce negative curvature (Suetsugu et al.,
2009). Unrelated proteins also use a similar strategy: the Brol domain has a
convex membrane-binding site and induces negative curvature and the COPII
adaptor complex Sec23*34 has a concave membrane-binding surface that
stabilizes positive curvature initiated by Sari (Bi et al., 2002; Kim et al., 2005).
While sequence analysis of Nups has not revealed homology to these types of
domains, clearly a number of systems have evolved to utilize similar structures. It
will be very interesting to see if a Nup or complex of Nups will similarly present a
curved lipid-binding surface to impart and/or stabilize curvature.
Additionally, the lipid composition at the pore membrane could have effects on
membrane organization and structure and its contributions remain unexplored.
147
As the architecture of the NPC becomes clear, significant interplay between
different mechanisms of membrane deformation and stabilization is likely to be
revealed.
Summary
Structural insight in to the NPC has progressed rapidly over the past several
years. While a complete atomic level structure is still far in the future, work
described in this thesis has allowed us to produce a model for the NPC
architecture that is lattice-like based on an evolutionary relationship with the
COPIl vesicle coat. Importantly, this insight and a working model of the NPC
lattice allows for the generation of a number of specific hypotheses for the
manner of incorporation and function of uncharacterized Nups and the NPC
assembly as a whole. Much exciting work on the path to a full structural
description of the NPC is clearly still to come.
148
Appendix
A lattice model for the nuclear pore complex
This appendix was previously published as Brohawn, S.G. & Schwartz, T.U. A
lattice model for the nuclear pore complex. Communicative and Integrative
Biology 2, 205-207 (2009).
S.G.B. prepared the figure and wrote the manuscript; T.U.S. advised on all
aspects of the project and wrote the manuscript.
149
Abstract
The nuclear pore complex (NPC) is one of the largest protein machines in the
cell and forms the sole conduit for nucleocytoplasmic transport in eukaryotes.
The NPC is composed of an eightfold radially symmetric scaffold of architectural
proteins that anchor a set of phenylalanine-glycine (FG) repeat proteins that form
the transport barrier. As a step toward elucidating the molecular architecture of
the NPC, we solved the structure of nucleoporin 85 (Nup85) in complex with
Seh1, a module in the heptameric Nup84 or Y subcomplex. We define a new
tripartite protein element, the ancestral coatomer element ACE1, which Nup85
specifically shares with several other nucleoporins and vesicle coat proteins. We
predicted and verified functional sites on nucleoporin ACE1 members based on
analogy to ACE1 interactions that propagate the COPII vesicle coat. Thus, we
provide the first experimental evidence for evolution of the NPC and vesicle coats
from a common ancestor. We propose that the NPC structural scaffold, like
vesicle coats, is a polygonal network composed of vertex and edge elements that
forms a molecular lattice upon which additional nucleoporins assemble. Here we
further discuss our findings and elaborate on our lattice model of the nuclear pore
complex.
Discussion
All nucleocytoplasmic transport in a cell proceeds through nuclear pore
complexes (NPCs). NPCs are composed of an eight-fold radially symmetric
structural scaffold that anchors a group of FG-repeat-containing proteins that
form the transport barrier (D'Angelo and Hetzer, 2008; Tran and Wente, 2006;
Weis, 2003). Elucidating the three dimensional structure of the NPC is critical for
understanding its roles in nucleocytoplasmic transport and cellular homeostasis.
A path towards an atomic resolution structure of the 40-60 MDa NPC is made
possible by the realization that the NPC is a modular assembly (Schwartz, 2005).
The NPC is composed of -30 proteins (Nups) that are arranged into distinct
subcomplexes, each present in multiple copies (Rout et al., 2000). The structural
scaffold contains the most stably attached nups and comprises two
150
subcomplexes in yeast: the heptameric Y complex (composed of Nup133,
Nup84, Nup145C, Sec13, Nup85, Seh1, and Nup120) and the heteromeric Nic96
subcomplex (likely composed of Nic96, Nup192, Nup188, Nup157/170, Nup59,
and Nup53) (Rabut et al., 2004).
In an attempt to better understand the architecture of the NPC, we solved the
crystal structure of Nup85 in complex with Seh1 (Brohawn et al., 2008). Nup85
interacts with Seh1 via insertion of an N-terminal insertion blade into the open 6bladed p-propeller of Seh1. The remainder of Nup85 forms a uniquely arrayed Jshaped a-helical block with two distinct units we term "crown" and "trunk". The
fold is notably different from the regular a-helical stack that was predicted (Devos
et al., 2004; Devos et al., 2006).
We found that this fold is shared in four other proteins of known structure: the
nuc~leoporins Nup145C, Nup84, and Nic96 and the COPIl vesicle coatomer
Sec3l (Boehmer et al., 2008; Fath et al., 2007; Hsia et al., 2007; Jeudy and
Schwartz, 2007). While a related architecture between the NPC and vesicle
coats has been proposed based on similar fold composition (Devos et al., 2004),
we provide the first experimental and structural evidence of a common ancestry
(Brohawn et al., 2008). Comparison of the structures shows a shared core
composed of three modules: the crown, trunk and C-terminal tail. We termed this
tripartite fold the ancestral coatomer element 1 (ACE1). While the overall
organization and topology is identical, there are significant differences in the
relative orientation of the modules between members. This suggests the
boundaries between ACE1 modules may function as hinges. This relationship
was not previously predicted due to low sequence conservation and was initially
obscured at the structural level by differences in relative orientations of the
modules (Hsia et al., 2007).
We used characterized interactions of ACE1 proteins to predict functional sites
on other members. First, in COPII vesicle coats, Sec31-Sec13 dimers form edge
151
elements through Sec3l crown-crown homodimerization (Figure A.1A) (Fath et
al., 2007). We predicted and demonstrated that Nup145C likewise interacts
crown-crown with its binding partner in the Y complex, Nup84. Second, we
predicted and verified that Nup85 and Nup145C tails interact with Nup120 as the
Nup84 tail module interacts with Nup133 (Boehmer et al., 2008). These and
other data were used to construct an improved model of the Y complex (Figure
A.1B).
We propose that the nuclear pore complex scaffold has a lattice structure
assembled from vertex and edge elements similar in principle to vesicle coats.
We can envision at least two alternative models for this lattice. Inthe first
(Figure A. 1B(i)), two rings of the Y complex sandwich an inner ring of the Nic96
subcomplex. This arrangement would generate a scaffold of -50 nm diameter,
consistent with the observed pore size in yeast (Kiseleva et al., 2004; Yang et al.,
1998). Alternatively (Figure A. 1B(ii)), the scaffold may consist of two stacked
rings of the Y complex without an intervening Nic96 subcomplex ring, which may
be sufficient to traverse the -30-50 nm pore height. Uncertainty about the exact
arrangement arises from still incompletely understood stoichiometries of
components and limited information about overall NPC size. Future clarification
of the connectivity between scaffold subcomplexes will additionally help to
discern the possible lattice arrangements. Homology to COPII coatomers
suggests ACE1-containing subcomplexes will be edge elements in the NPC
lattice. The nature of the vertex elements in the NPC is less clear, though it may
well also involve p-propeller-p-propeller interactions.
152
..........................................
.
...........
Sec13
Sec13
(i)
rng
Cytoplasmic
Nucleop
ng
xxxxxx
Cytoplasmic ring
v i
-20nm
a
inng
Figure A.1 - A lattice model of the NPC
Sec3l, Nup85, Nup145C, Nup84, and Nic96 ACE1 proteins are colored with
crowns blue, trunks orange, and tail modules green. Other protein folds are
shown in grey. (A) Schematic organization of the COPIl outer vesicle coat. On
the left, an edge element consisting of two Sec3l -Sec1 3 heterodimers is shown.
Two Sec3l molecules interact crown-crown. On the right, an entire COPIl
cuboctahedron coat composed of 24 edge elements is shown unwrapped and
laid flat (Fath et al., 2007). Vertex elements are formed where four Sec3l and
four Sec1 3 p-propellers interact. (B)Alternative organizations of the NPC lattice.
On the left, the Y complex is shown in schematic fashion illustrating how ACE1
interactions organize the Y-shaped structure. Nup145C and Nup84 also interact
crown-crown. On the right, the entire NPC structural scaffold is shown
unwrapped and laid flat. Two rings of the Y complex form the lattice of the NPC
scaffold either with an intervening ring of the Nic96 subcomplex (i) or alone (ii).
Both the identity and organization of the vertex elements and the Nic96
subcomplex in the pore lattice are unknown and are shown half-transparent. The
presented organization is not meant to predict relative positions of proteins or the
structure per se, but rather emphasizes the principally similar lattice organization
of NPCs and vesicle coats.
153
Our lattice model of the NPC prompts a number of additional potential parallels to
vesicle coats. First, vesicle coatomers do not directly contact membranes, but
use adaptor protein complexes to span the -8 nm gap and recruit cargo (Owen
et al., 2004). Consistently, a -8 nm gap has been observed between the
structural scaffold of the NPC and the nuclear membrane (Beck et al., 2007).
Conceivably other nups fill corresponding adaptor complex roles by linking the
lattice to transmembrane nups, or transmembrane nups could act directly as
adaptors. Second, COPII vesicle coats size flexibility is made possible largely by
hinges at coat vertices (Stagg et al., 2008). It may be that analogous hinges as
well as those between ACE1 modules in the NPC lattice confer plasticity that
may be used for pore dilation or NPC (dis)assembly.
Recent work has produced two conflicting models of the molecular organization
of the NPC. A computationally generated model that integrates a wealth of
localization, interaction, and other primary data similarly places the Y complex in
two peripheral NPC rings flanking an inner ring composed of the Nic96
subcomplex (Alber et al., 2007a; Alber et al., 2007b). In contrast, a model based
on crystal packing interactions in Nupl45C-Secl3 places the Y complex in four
stacked rings organized by hetero-octameric poles of Nup145C-Sec13 and
Nup85*Sehl units (Hsia et al., 2007). A tube of 32 Y complexes was proposed to
envelope inner cylinders of the Nic96 subcomplex and FG Nups generating a
"concentric cylinder" model of the NPC (Debler et al., 2008; Hsia et al., 2007).
Our model is incompatible with the "concentric cylinder" model for the NPC.
Specifically, the demonstrated crown-crown interaction between Nup84 and
Nup145C overlaps with Nup145C crystal contacts necessary for the propagation
of the models hetero-octameric poles. Exposed hydrophobic surfaces tend to
form crystal-packing contacts; whether or not they are physiologically relevant
needs to be addressed by additional experiments (Kobe et al., 2008). Careful
analysis of packing interactions in crystals can unveil biologically important
protein interfaces, especially if the crystallized proteins are part of a higher-order
154
assembly in vivo, as nups are. Crystal contacts with at least some hydrophobic
character are also observed in the structures of Nup85-Sehl, Nup1 07-Nupl 33
and Nic96 (Boehmer et al., 2008; Brohawn et al., 2008; Jeudy and Schwartz,
2007; Schrader et al., 2008b), which are all fragments of larger assemblies.
While some of these interactions can likely be ruled out as crystal artifacts, i.e.
because they involve surfaces created by the use of truncated proteins that
would otherwise be buried in the hydrophobic core of the protein, others may be
indicative of real functional sites. Weak interactions observed in crystals may
point to inter-subcomplex contact areas that are important for self-assembly and
to date have not been observed in solution.
We have provided the first structural evidence of a common ancestry of vesicle
coats and the nuclear pore complex and provide a lattice model of the NPC
based on this commonality. Our lattice model is generally consistent with the
computational model of the NPC (Alber et al., 2007b), though the absence of
additional structural knowledge precludes a detailed comparison. Our model
provides a framework upon which further structural and cell biological studies can
be placed in an effort to more fully understand the assembly principles and
function of the NPC.
Acknowledgements
This work was supported by NIH grant GM77537 (T.U.S.), a Pew Scholar Award
(T.U.S.), a Koch Fellowship Award (S.G.B.), and a Vertex Scholarship (S.G.B.).
155
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